O-linked glycosylation of peptides

ABSTRACT

The present invention provides polypeptides that include an O-linked glycosylation site that is not present in the wild-type peptide. The polypeptides of the invention include glycoconjugates in which a species such as a water-soluble polymer, a therapeutic agent of a biomolecule is covalently linked through an intact O-linked glycosyl residue to the polypeptide. Also provided are methods of making the peptides of the invention and methods, pharmaceutical compositions containing the peptides and methods of treating, ameliorating or preventing diseased in mammals by administering an amount of a peptide of the invention sufficient to achieve the desired response.

CROSS-REFERENCES TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 11/033,365, filed Jan. 10, 2005, which is related to U.S.Provisional Patent Application No. 60/535,284, filed Jan. 8, 2004, U.S.Provisional Patent Application No. 60/544,411, filed Feb. 12, 2004; U.S.Provisional Patent Application No. 60/546,631, filed Feb. 20, 2004; U.S.Provisional Patent Application No. 60/555,813, filed Mar. 23, 2004; U.S.Provisional Patent Application No. 60/570,891, filed May 12, 2004; eachof which are incorporated herein by reference in their entirety for allpurposes.

FIELD OF THE INVENTION

The present invention relates to O-linked glycosylated glycopeptides,particularly therapeutic peptides and peptide mutants that includeO-linked glycosylation sites not present in the wild-type peptide.

The administration of glycosylated and non-glycosylated peptides forengendering a particular physiological response is well known in themedicinal arts. For example, both purified and recombinant hGH are usedfor treating conditions and diseases due to hGH deficiency, e.g.,dwarfism in children, interferon has known antiviral activity andgranulocyte colony stimulating factor stimulates the production of whiteblood cells.

A principal factor that has limited the use of therapeutic peptides isthe difficulty inherent in engineering an expression system to express apeptide having the glycosylation pattern of the wild-type peptide. As isknown in the art, improperly or incompletely glycosylated peptides canbe immunogenic, leading to neutralization of the peptide and/or leadingto the development of an allergic response. Other deficiencies ofrecombinantly produced glycopeptides include suboptimal potency andrapid clearance rates.

One approach to solving the problems inherent in the production ofglycosylated peptide therapeutics has been to modify the peptides invitro after they are expressed. Post-expression in vitro modificationhas been used to both modify of glycan structures and introduce ofglycans at novel sites. A comprehensive toolbox of recombinanteukaryotic glycosyltransferases has become available, making in vitroenzymatic synthesis of mammalian glycoconjugates with custom designedglycosylation patterns and glycosyl structures possible. See, forexample, U.S. Pat. Nos. 5,876,980; 6,030,815; 5,728,554; 5,922,577; andWO/9831826; US2003180835; and WO 03/031464.

In addition to manipulating the structure of glycosyl groups onpolypeptides, glycopeptides can be prepared with one or morenon-saccharide modifying groups, such as water soluble polymers. Anexemplary polymer that has been conjugated to peptides is poly(ethyleneglycol) (“PEG”). The use of PEG to derivative peptide therapeutics hasbeen demonstrated to reduce the immunogenicity of the peptides. Forexample, U.S. Pat. No. 4,179,337 (Davis et al.) disclosesnon-immunogenic polypeptides such as enzymes and peptide hormonescoupled to polyethylene glycol (PEG) or polypropylene glycol. Inaddition to reduced immunogenicity, the clearance time in circulation isprolonged due to the increased size of the PEG-conjugate of thepolypeptides in question.

The principal mode of attachment of PEG, and its derivatives, topeptides is a non-specific bonding through a peptide amino acid residue(see e.g., U.S. Pat. No. 4,088,538 U.S. Pat. No. 4,496,689, U.S. Pat.No. 4,414,147, U.S. Pat. No. 4,055,635, and PCT WO 87/00056). Anothermode of attaching PEG to peptides is through the non-specific oxidationof glycosyl residues on a glycopeptide (see e.g., WO 94/05332).

In these non-specific methods, poly(ethyleneglycol) is added in arandom, non-specific manner to reactive residues on a peptide backbone.Of course, random addition of PEG molecules has its drawbacks, includinga lack of homogeneity of the final product, and the possibility forreduction in the biological or enzymatic activity of the peptide.Therefore, for the production of therapeutic peptides, a derivitizationstrategy that results in the formation of a specifically labeled,readily characterizable, essentially homogeneous product is superior.

Specifically labeled, homogeneous peptide therapeutics can be producedin vitro through the action of enzymes. Unlike the typical non-specificmethods for attaching a synthetic polymer or other label to a peptide,enzyme-based syntheses have the advantages of regioselectivity andstereoselectivity. Two principal classes of enzymes for use in thesynthesis of labeled peptides are glycosyltransferases (e.g.,sialyltransferases, oligosaccharyltransferases,N-acetylglucosaminyltransferases), and glycosidases. These enzymes canbe used for the specific attachment of sugars which can be subsequentlymodified to comprise a therapeutic moiety. Alternatively,glycosyltransferases and modified glycosidases can be used to directlytransfer modified sugars to a peptide backbone (see e.g., U.S. Pat. No.6,399,336, and U.S. Patent Application Publications 20030040037,20040132640, 20040137557, 20040126838, and 20040142856, each of whichare incorporated by reference herein). Methods combining both chemicaland enzymatic synthetic elements are also known (see e.g., Yamamoto etal. Carbohydr. Res. 305: 415-422 (1998) and U.S. Patent ApplicationPublication 20040137557 which is incorporated herein by reference).

Carbohydrates are attached to glycopeptides in several ways of whichN-linked to asparagine and mucin-type O-linked to serine and threonineare the most relevant for recombinant glycoprotein therapeutics.Unfortunately, not all polypeptide comprise an N- or O-linkedglycosylation site as part of their primary amino acid sequence. Inother cases an existing glycosylation site may be inconvenient for theattachment of a modifying group (e.g., a water-soluble orwater-insoluble polymers, therapeutic moieties, and or biomolecules) tothe polypeptide, or attachment of such moieties at that site may causean undesirable decrease in biological activity of the polypeptide. Thusthere is a need in the art for methods that permit both the precisecreation of glycosylation sites and the ability to precisely direct themodification of those sites.

SUMMARY OF THE INVENTION

It is a discovery of the present invention that enzymaticglycoconjugation reactions can be specifically targeted to O-linkedglycosylation sites and to glycosyl residues that are attached toO-linked glycosylation sites. The targeted O-linked glycosylation sitescan be sites native to a wild-type peptide or, alternatively, they canbe introduced into a peptide by mutation. Accordingly, the presentinvention provides polypeptides comprising mutated sites suitable forO-linked glycosylation and pharmaceutical compositions thereof. Inaddition, the present invention provides methods of making suchpolypeptides and using such polypeptides and/or pharmaceuticalcompositions thereof for therapeutic treatments.

Thus, in a first aspect, the invention provides an isolated polypeptidecomprising a mutant peptide sequence, wherein the mutant peptidesequence encodes an O-linked glycosylation site that does not exist inthe corresponding wild-type polypeptide.

In one embodiment, the isolated polypeptide is a G-CSF polypeptide.

In one embodiment, the G-CSF polypeptide comprises a mutant peptidesequence with the formula of M¹X_(n)TPLGP or M¹B_(o)PZ_(m)X_(n)TPLGP. Inthis embodiment, the superscript, 1, denotes the first position of theamino acid sequence of the wild-type G-CSF sequence (SEQ ID NO:3), thesubscripts n and m are integers selected from 0 to 3, and at least oneof X and B is threonine or serine, and when more than one of X and B isthreonine or serine, the identity of these moieties is independentlyselected. Also in this embodiment, Z is selected from glutamate, anyuncharged amino acid or dipeptide combination including MQ, GQ, and MV.In another embodiment, the G-CSF polypeptide comprises a mutant peptidesequence selected from the sequences consisting of MVTPLGP, MQTPLGP,MIATPLGP, MATPLGP, MPTQGAMPLGP, MVQTPLGP, MQSTPLGP, MGQTPLGP,MAPTSSSPLGP, and MAPTPLGPA.

In another embodiment, the G-CSF polypeptide comprises a mutant peptidesequence with the formula of M¹TPXBO_(r)P. In this embodiment thesuperscript, 1, denotes the first position of the amino acid sequence ofthe wild-type G-CSF sequence (SEQ ID NO:3), and the subscript r is aninteger selected from 0 to 3, and at least one of X, B and O isthreonine or serine, and when more than one of X, B and O is threonineor serine, the identity of these moieties is independently selected. Inanother embodiment, the G-CSF polypeptide comprises a mutant peptidesequence selected from the sequences consisting of: MTPTLGP, MTPTQLGP,MTPTSLGP, MTPTQGP, MTPTSSP, M¹TPQTP, M¹TPTGP, M¹TPLTP, M¹TPNTGP, MTPLGP,M¹TPVTP, M¹TPMVTP, and MT¹P²TQGL³G⁴P⁵A⁶S⁷.

In another embodiment, the G-CSF polypeptide comprises a mutant peptidesequence with the formula of LGX⁵³B_(o)LGI, wherein the superscriptdenotes the position of the amino acid in the wild type G-CSF amino acidsequence, and X is histidine, serine, arginine, glutamic acid ortyrosine, and B is either threonine or serine, and o is an integer from0 to 3.

In another embodiment, the G-CSF polypeptide comprises a mutant peptidesequence selected from the sequences consisting of: LGHTLGI, LGSSLGI,LGYSLGI, LGESLGI, and LGSTLGI.

In another embodiment, the G-CSF polypeptide comprises a mutant peptidesequence with the formula of P¹²⁹Z_(m)J_(q)O_(r)X_(n)PT wherein thesuperscript denotes the position of the amino acid in the wild typeG-CSF amino acid sequence, and Z, J, O and X are independently selectedfrom threonine or serine, and m, q, r, and n are integers independentlyselected from 0 to 3. In another embodiment, the G-CSF polypeptidecomprises a mutant peptide sequence selected from the sequencesconsisting of: P¹²⁹ATQPT, P¹²⁹TLGPT, P¹²⁹TQGPT, P¹²⁹TSSPT, P¹²⁹TQGAPT,P¹²⁹NTGPT, PALQPTQT, P¹²⁹ALTPT, P¹²⁹MVTPT, P¹²⁹ASSTPT, P¹²⁹TTQP,P¹²⁹NTLP, P¹²⁹ TLQP, MAP¹²⁹ATQPTQGAM, and MP¹²⁹ATTQPTQGAM.

In another embodiment, the G-CSF polypeptide comprises a mutant peptidesequence with the formula of P¹²⁹Z_(m)U_(s)J_(q)P⁶¹O_(r)X_(n)BOC whereinthe superscript denotes the position of the amino acid in the wild typeG-CSF amino acid sequence, and at least one of Z, J, O, and U isselected from threonine or serine, and when more than one of Z, J, O andU is threonine or serine, each is independently selected, X and B areany uncharged amino acid or glutamate, and m, s, q, r, n, and o areintegers independently selected from 0 to 3. In another embodiment theG-CSF polypeptide comprises a mutant peptide sequence selected from thesequences consisting of: P⁶¹TSSC, P⁶¹TSSAC, LGIPTA P⁶¹LSSC, LGIPTQP⁶¹LSSC, LGIPTQG P⁶¹LSSC, LGIPQT P⁶¹LSSC, LGIPTS P⁶¹LSSC, LGIPTSP⁶¹LSSC, LGIPTQP⁶¹LSSC, LGTPWAP⁶¹ LSSC, LGTPFA P⁶¹LSSC, P⁶¹FTP, andSLGAP⁵⁸TAP⁶¹LSS.

In another embodiment the G-CSF polypeptide comprises a mutant peptidesequence with the formula of Ø_(a)G_(p)J_(q)O_(r)P¹⁷⁵X_(n)B_(o)Z_(m)U_(s)Ψ_(t) wherein the superscript denotes the positionof the amino acid in SEQ ID NO:3, and at least one of Z, U, O, J, G, Ø,B and X is threonine or serine and when more than one of Z, U, O, J, G,Ø, B and X are threonine or serine, they are independently selected. Øis optionally R, and G is optionally H. The symbol Ψ represents anyuncharged amino acid residue or glutamate, and a, p, q, r, n, o, m, s,and t are integers independently selected from 0 to 3. In anotherembodiment the G-CSF polypeptide comprises a mutant peptide sequenceselected from the sequences consisting of: RHLAQTP¹⁷⁵, RHLAGQTP¹⁷⁵,QP¹⁷⁵TQGAMP, RHLAQTP¹⁷⁵AM, QP¹⁷⁵TSSAP, QP¹⁷⁵TSSAP, QP¹⁷⁵TQGAMP,QP¹⁷⁵TQGAM, QP¹⁷⁵TQGA, QP¹⁷⁵TVM, QP¹⁷⁵NTGP, and QP¹⁷⁵QTLP.

In another embodiment the G-CSF polypeptide comprises a mutant peptidesequence selected from the sequences P¹³³TQTAMP¹³⁹, P¹³³TQGTMP,P¹³³TQGTNP, P¹³³TQGTLP, and PALQP¹³³TQTAMPA.

In another embodiment, the isolated polypeptide is an hGH polypeptide.

In one embodiment, the hGH polypeptide comprises a mutant peptidesequence with the formula of P¹³³JXBOZUK¹⁴⁰QTYS, wherein superscriptsdenote the position of the amino acid in (SEQ ID NO:20); and J isselected from threonine and arginine; X is selected from alanine,glutamine, isoleucine, and threonine; B is selected from glycine,alanine, leucine, valine, asparagine, glutamine, and threonine; O isselected from tyrosine, serine, alanine, and threonine; and Z isselected from isoleucine and methionine; and U is selected fromphenylalanine and proline. In another embodiment, the hGH polypeptidecomprises a mutant peptide sequence selected from the sequencesconsisting of: PTTGQIFK, PTTAQIFK, PTTLQIFK, PTTLYVFK, PTTVQIFK,PTTVSIFK, PTTNQIFK, PTTQQIFK, PTATQIFK, PTQGQIFK, PTQGAIFK, PTQGAMFK,PTIGQIFK, PTINQIFK, PTINTIFK, PTILQIFK, PTIVQIFK, PTIQQIFK, PTIAQIFK,P¹³³TTTQIFK¹⁴⁰QTYS, and P¹³³TQGAMPK¹⁴⁰QTYS.

In another embodiment, the hGH polypeptide comprises a mutant peptidesequence with the formula of P¹³³RTGQIPTQBYS wherein superscripts denotethe position of the amino acid in SEQ ID NO:20; and B is selected fromalanine and threonine. In another embodiment, the hGH polypeptidecomprises a mutant peptide sequence selected from the sequencesconsisting of: PRTGQIPTQTYS and PRTGQIPTQAYS.

In another embodiment, the hGH polypeptide comprises a mutant peptidesequence with the formula of L¹²⁸XTBOP¹³³UTG wherein superscripts denotethe position of the amino acid in SEQ ID NO:20; and X is selected fromglutamic acid, valine and alanine; B is selected from glutamine,glutamic acid, and glycine; O is selected from serine and threonine; andU is selected from arginine, serine, alanine and leucine. In anotherembodiment, the hGH polypeptide comprises a mutant peptide sequenceselected from the sequences consisting of: LETQSP¹³³RTG, LETQSP¹³³STG,LETQSP¹³³ATG, LETQSP¹³³LTG, LETETP¹³³R, LETETP¹³³A, LVTQSP¹³³RTG,LVTETP¹³³RTG, LVTETP¹³³ATG, and LATGSP¹³³RTG.

In another embodiment the hGH polypeptide comprises a mutant peptidesequence with the formula of M¹BPTX_(n)Z_(m)OPLSRL wherein thesuperscript 1, denotes the position of the amino acid in SEQ ID NO:19;and B is selected from phenylalanine, valine and alanine or acombination thereof, X is selected from glutamate, valine and proline Zis threonine; O is selected from leucine and isoleucine; and when X isproline, Z is threonine; and wherein n and m are integers selected from0 and 2. In another embodiment, the hGH polypeptide comprises a mutantpeptide sequence selected from the sequences consisting of: M¹FPTEIPLSRL, M¹FPTV LPLSRL, and M¹APTPTIPLSRL.

In still another embodiment the hGH polypeptide comprises the followingmutant peptide sequence: M¹VTPTIPLSRL.

In still another embodiment the hGH polypeptide comprises a mutantpeptide sequence selected from M¹APTSSPTIPL⁷SR⁹ and DGSP¹³³NTGQIFK¹⁴⁰.

In another embodiment the isolated polypeptide is an IFN alphapolypeptide.

In one embodiment, the INF alpha polypeptide has a peptide sequencecomprising a mutant amino acid sequence, and the peptide sequencecorresponds to a region of INF alpha 2 having a sequence as shown in SEQNO:22, and wherein the mutant amino acid sequence contains a mutation ata position corresponding to T¹⁰⁶ of INF alpha 2. In another embodimentthe IFN alpha polypeptide is selected from the group consisting of IFNalpha, IFN alpha 4, IFN alpha 5, IFN alpha 6, IFN alpha 7, IFN alpha 8,IFN alpha 10, IFN alpha 14, IFN alpha 16, IFN alpha 17, and IFN alpha21. In yet another embodiment, the IFN alpha polypeptide is an IFN alphapolypeptide comprising a mutant amino acid sequence selected from thegroup consisting of: ⁹⁹CVMQEERVTETPLMNADSIL¹¹⁸,⁹⁹CVMQEEGVTETPLMNADSIL¹¹⁸, and ⁹⁹CVMQGVGVTETPLMNADSIL¹¹⁸. In stillanother embodiment, the IFN alpha polypeptide is an IFN alpha 4polypeptide comprising a mutant amino acid sequence selected from thegroup consisting of: ⁹⁹CVIQEVGVTETPLMNVDSIL¹¹⁸, and⁹⁹CVIQGVGVTETPLMKEDSIL¹¹⁸. In another embodiment, the IFN alphapolypeptide is an IFN alpha 5 polypeptide comprising a mutant amino acidsequence selected from the group consisting of:⁹⁹CMMQEVGVTDTPLMNVDSIL¹¹⁸, ⁹⁹CMMQEVGVTETPLMNVDSIL¹¹⁸ and⁹⁹CMMQGVGVTDTPLMNVDSIL¹¹⁸. In an another embodiment, the IFN alphapolypeptide is an IFN alpha 6 polypeptide comprising a mutant amino acidsequence selected from the group consisting of:⁹⁹CVMQEVWVTGTPLMNEDSIL¹¹⁸, ⁹⁹CVMQEVGVTGTPLMNEDSIL¹¹⁸, and⁹⁹CVMQGVGVTETPLMNEDSIL¹¹⁸. In yet an another embodiment, the IFN alphapolypeptide is an IFN alpha 7 polypeptide comprising a mutant amino acidsequence selected from the group consisting of:⁹⁹CVIQEVGVTETPLMNEDFIL¹¹⁸, and ⁹⁹CVIQGVGVTETPLMNEDFIL¹¹⁸. In stillanother embodiment, the IFN alpha polypeptide is an IFN alpha 8polypeptide comprising a mutant amino acid sequence selected from thegroup consisting of: ⁹⁹CVMQEVGVTESPLMYEDSIL¹¹⁸, and⁹⁹CVMQGVGVTESPLMYEDSIL¹¹⁸. In another embodiment, the IFN alphapolypeptide is an IFN alpha 10 polypeptide comprising a mutant aminoacid sequence selected from the group consisting of:⁹⁹CVIQEVGVTETPLMNEDSIL¹¹⁸, and ⁹⁹CVIQGVGVTETPLMNEDSIL¹¹⁸. In anotherembodiment, the IFN alpha polypeptide is an IFN alpha 14 polypeptidecomprising a mutant amino acid sequence selected from the groupconsisting of: ⁹⁹CVIQEVGVTETPLMNEDSIL¹¹⁸, and ⁹⁹CVIQGVGVTETPLMNEDSIL¹¹⁸.In another embodiment, the IFN alpha polypeptide is an IFN alpha 16polypeptide comprising a mutant amino acid sequence selected from thegroup consisting of: ⁹⁹CVTQEVGVTEIPLMNEDSIL¹¹⁸,⁹⁹CVTQEVGVTETPLMNEDSIL¹¹⁸, and ⁹⁹CVTQGVGVTETPLMNEDSIL¹¹⁸. In stillanother embodiment, the IFN alpha polypeptide is an IFN alpha 17polypeptide comprising a mutant amino acid sequence selected from thegroup consisting of: ⁹⁹CVIQEVGMTETPLMNEDSIL¹¹⁸,⁹⁹CVIQEVGVTETPLMNEDSIL¹¹⁸, and ⁹⁹CVIQGVGMTETPLMNEDSIL¹¹⁸. In one moreembodiment, the IFN alpha polypeptide is an IFN alpha 21 polypeptidecomprising a mutant amino acid sequence selected from the groupconsisting of: ⁹⁹CVIQEVGVTETPLMNVDSIL¹¹⁸, and ⁹⁹CVIQGVGVTETPLMNVDSIL¹¹⁸.

In a second aspect, the invention provides an isolated nucleic acidencoding a polypeptide comprising a mutant peptide sequence, wherein themutant peptide sequence encodes an O-linked glycosylation site that doesnot exist in the corresponding wild-type polypeptide. In one embodimentthe nucleic acid encoding a polypeptide comprising a mutant peptidesequence is comprised within an expression cassette. In another relatedembodiment, the present invention provides a cell comprises the nucleicacid of the present invention.

In a third aspect, the isolated polypeptide comprising a mutant peptidesequence, that encodes an O-linked glycosylation site that not existingin the corresponding wild-type polypeptide, has a formula selected from:

wherein AA is an amino acid side chain that comprises a hydroxyl moietythat is within the mutant polypeptide sequence; and X is a modifyinggroup or a saccharyl moiety. In one embodiment X comprises a groupselected from sialyl, galactosyl and Gal-Sia moieties, wherein at leastone of said sialyl, galactosyl and Gal-Sia comprises a modifying group.

In another embodiment X comprises the moiety:

wherein D is a member selected from —OH and R¹-L-HN—; G is a memberselected from R¹-L- and —C(O)(C₁-C₆)alkyl; R¹ is a moiety comprising amember selected a moiety comprising a straight-chain or branchedpoly(ethylene glycol) residue; and L is a linker which is a memberselected from a bond, substituted or unsubstituted alkyl and substitutedor unsubstituted heteroalkyl, such that when D is OH, G is R¹-L-, andwhen G is —C(O)(C₁-C₆)alkyl, D is R¹-L-NH—.

In another embodiment X comprises the structure:

in which L is a substituted or unsubstituted alkyl or substituted orunsubstituted heteroalkyl group; and n is selected from the integersfrom 0 to about 500.

In another embodiment, X comprises the structure:

in which s is selected from the integers from 0 to 20.

In a fourth aspect the invention provides a method for making aglycoconjugate of an isolated polypeptide comprising a mutant peptidesequence encoding an O-linked glycosylation site that does not existingin the corresponding wild-type polypeptide, comprising the steps of:

(a) recombinantly producing the mutant polypeptide, and(b) enzymatically glycosylating the mutant polypeptide with a modifiedsugar at said O-linked glycosylation site.

In a fifth aspect the invention provides a pharmaceutical composition ofan isolated polypeptide comprising a mutant peptide sequence, whereinthe mutant peptide sequence encodes an O-linked glycosylation site thatdoes not exist in the corresponding wild-type polypeptide.

In one embodiment the pharmaceutical composition comprises an effectiveamount of a G-CSF polypeptide of the invention glycoconjugated with amodified sugar. In a related embodiment, the modified sugar is modifiedwith a member selected from poly(ethylene glycol) andmethoxy-poly(ethylene glycol) (m-PEG).

In another embodiment the pharmaceutical composition comprises aneffective amount of an hGH polypeptide of the invention glycoconjugatedwith a modified sugar. In a related embodiment, the modified sugar ismodified with a member selected from poly(ethylene glycol) andmethoxy-poly(ethylene glycol) (m-PEG).

In another embodiment the pharmaceutical composition comprises aneffective amount of an granulocyte macrophage colony stimulating factorpolypeptide of the invention glycoconjugated with a modified sugar. In arelated embodiment, the modified sugar is modified with a memberselected from poly(ethylene glycol) and methoxy-poly(ethylene glycol)(m-PEG).

In another embodiment the pharmaceutical composition comprises aneffective amount of an IFN alpha polypeptide of the inventionglycoconjugated with a modified sugar. In a related embodiment, themodified sugar is modified with a member selected from poly(ethyleneglycol) and methoxy-poly(ethylene glycol) (m-PEG).

In a sixth aspect the invention provides a method of providing therapyto a subject in need of said therapy, wherein the method comprises,administering to said subject an effective amount a pharmaceuticalcomposition of the invention. In one embodiment, the therapy provided isG-CSF therapy. In another embodiment the therapy provided is granulocytemacrophage colony stimulating factor therapy. In another embodiment thetherapy provided is interferon alpha therapy. In still anotherembodiment the therapy provided is Growth Hormone therapy.

Additional aspects, advantages and objects of the present invention willbe apparent from the detailed description that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a plot of absorbance vs. GCSF concentration for unmodifiedG-CSF and glyco-PEG-ylated analogues in a NSF-60 cell proliferationassay.

FIG. 2 is a plot of counts per minute (CPM) vs. time for a ratpharmacokinetic study using radioiodinated G-CSF and glycol-PEG-latedderivatives thereof.

FIG. 3 is a plot of μg/mL G-CSF in blood vs. time (h) for a ratpharmacokinetic study using radioiodinated G-CSF and glycol-PEG-latedderivatives thereof.

FIG. 4 is a plot showing the induction of white blood cells in miceusing unmodified G-CSF and chemically- and glyco-PEG-ylated G-CSF.

FIG. 5 is a plot of the results of an aggregation assay followingradioiodination with the Bolton-Hunter reagent.

FIG. 6 is a plot of the results of an accelerated stability study ofglyco-PEG-ylated G-CSF.

FIG. 7 is an expanded view of FIG. 6.

FIG. 8 is a plot of the results of a rat IV pK Study using the BoltonHunter radiolabeling process (precipitated plasma protein).

FIG. 9 is a plot of the results of a rat IV pK Study using unlabeledG-CSF, chemically- and glyco-PEG-ylated G-CSF detected by ELISA.

FIG. 10 shows representative sialyltransferases of use in the presentinvention.

DETAILED DESCRIPTION OF THE INVENTION Abbreviations

PEG, poly(ethyleneglycol); m-PEG, methoxy-poly(ethylene glycol); PPG,poly(propyleneglycol); m-PPG, methoxy-poly(propylene glycol); Fuc,fucosyl; Gal, galactosyl; GalNAc, N-acetylgalactosaminyl; Glc, glucosyl;GlcNAc, N-acetylglucosaminyl; Man, mannosyl; ManAc, mannosaminylacetate; Sia, sialic acid; and NeuAc, N-acetylneuraminyl.

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereingenerally have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs. Generally,the nomenclature used herein and the laboratory procedures in cellculture, molecular genetics, organic chemistry and nucleic acidchemistry and hybridization are those well known and commonly employedin the art. Standard techniques are used for nucleic acid and peptidesynthesis. The techniques and procedures are generally performedaccording to conventional methods in the art and various generalreferences (see generally, Sambrook et al. M OLECULAR CLONING: ALABORATORY MANUAL, 2d ed. (1989) Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., which is incorporated herein by reference),which are provided throughout this document. The nomenclature usedherein and the laboratory procedures in analytical chemistry, andorganic synthetic described below are those well known and commonlyemployed in the art. Standard techniques, or modifications thereof, areused for chemical syntheses and chemical analyses.

All oligosaccharides described herein are described with the name orabbreviation for the non-reducing saccharide (i.e., Gal), followed bythe configuration of the glycosidic bond (α or β), the ring bond (1 or2), the ring position of the reducing saccharide involved in the bond(2, 3, 4, 6 or 8), and then the name or abbreviation of the reducingsaccharide (i.e., GlcNAc). Each saccharide is preferably a pyranose. Fora review of standard glycobiology nomenclature see, Essentials ofGlycobiology Varki et al. eds. CSHL Press (1999).

Oligosaccharides are considered to have a reducing end and anon-reducing end, whether or not the saccharide at the reducing end isin fact a reducing sugar. In accordance with accepted nomenclature,oligosaccharides are depicted herein with the non-reducing end on theleft and the reducing end on the right.

The term “nucleic acid” or “polynucleotide” refers to deoxyribonucleicacids (DNA) or ribonucleic acids (RNA) and polymers thereof in eithersingle- or double-stranded form. Unless specifically limited, the termencompasses nucleic acids containing known analogues of naturalnucleotides that have similar binding properties as the referencenucleic acid and are metabolized in a manner similar to naturallyoccurring nucleotides. Unless otherwise indicated, a particular nucleicacid sequence also implicitly encompasses conservatively modifiedvariants thereof (e.g., degenerate codon substitutions), alleles,orthologs, SNPs, and complementary sequences as well as the sequenceexplicitly indicated. Specifically, degenerate codon substitutions maybe achieved by generating sequences in which the third position of oneor more selected (or all) codons is substituted with mixed-base and/ordeoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991);Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini etal., Mol. Cell. Probes 8:91-98 (1994)). The term nucleic acid is usedinterchangeably with gene, cDNA, and mRNA encoded by a gene.

The term “gene” means the segment of DNA involved in producing apolypeptide chain. It may include regions preceding and following thecoding region (leader and trailer) as well as intervening sequences(introns) between individual coding segments (exons).

The term “isolated,” when applied to a nucleic acid or protein, denotesthat the nucleic acid or protein is essentially free of other cellularcomponents with which it is associated in the natural state. It ispreferably in a homogeneous state although it can be in either a dry oraqueous solution. Purity and homogeneity are typically determined usinganalytical chemistry techniques such as polyacrylamide gelelectrophoresis or high performance liquid chromatography. A proteinthat is the predominant species present in a preparation issubstantially purified. In particular, an isolated gene is separatedfrom open reading frames that flank the gene and encode a protein otherthan the gene of interest. The term “purified” denotes that a nucleicacid or protein gives rise to essentially one band in an electrophoreticgel. Particularly, it means that the nucleic acid or protein is at least85% pure, more preferably at least 95% pure, and most preferably atleast 99% pure.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an a carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. “Amino acid mimetics” refers tochemical compounds having a structure that is different from the generalchemical structure of an amino acid, but that functions in a mannersimilar to a naturally occurring amino acid.

There are various known methods in the art that permit the incorporationof an unnatural amino acid derivative or analog into a polypeptide chainin a site-specific manner, see, e.g., WO 02/086075.

Amino acids may be referred to herein by either the commonly known threeletter symbols or by the one-letter symbols recommended by the IUPAC-IUBBiochemical Nomenclature Commission. Nucleotides, likewise, may bereferred to by their commonly accepted single-letter codes.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, “conservatively modified variants” refers to those nucleicacids that encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given protein. For instance, the codons GCA, GCC, GCGand GCU all encode the amino acid alanine. Thus, at every position wherean alanine is specified by a codon, the codon can be altered to any ofthe corresponding codons described without altering the encodedpolypeptide. Such nucleic acid variations are “silent variations,” whichare one species of conservatively modified variations. Every nucleicacid sequence herein that encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of skill willrecognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, each silent variation of a nucleic acidthat encodes a polypeptide is implicit in each described sequence.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art. Such conservatively modified variantsare in addition to and do not exclude polymorphic variants, interspecieshomologs, and alleles of the invention.

The following eight groups each contain amino acids that areconservative substitutions for one another:

1) Alanine (A), Glycine (G);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5)Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6)Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S),Threonine (T); and 8) Cysteine (C), Methionine (M)

(see, e.g., Creighton, Proteins (1984)).

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

In the present application, amino acid residues are numbered accordingto their relative positions from the most N-terminal residue, which isnumbered 1, in an unmodified wild-type polypeptide sequence.

“Peptide” refers to a polymer in which the monomers are amino acids andare joined together through amide bonds. Peptides of the presentinvention can vary in size, e.g., from two amino acids to hundreds orthousands of amino acids, which alternatively is referred to as apolypeptide. Additionally, unnatural amino acids, for example,β-alanine, phenylglycine and homoarginine are also included. Amino acidsthat are not gene-encoded may also be used in the present invention.Furthermore, amino acids that have been modified to include reactivegroups, glycosylation sites, polymers, therapeutic moieties,biomolecules and the like may also be used in the invention. All of theamino acids used in the present invention may be either the D- orL-isomer. The L-isomer is generally preferred. In addition, otherpeptidomimetics are also useful in the present invention. As usedherein, “peptide” refers to both glycosylated and unglycosylatedpeptides. Also included are peptides that are incompletely glycosylatedby a system that expresses the peptide. For a general review, see,Spatola, A. F., in CHEMISTRY AND BIOCHEMISTRY OF AMINO ACIDS, PEPTIDESAND PROTEINS, B. Weinstein, eds., Marcel Dekker, New York, p. 267(1983).

In the present application, amino acid residues are numbered accordingto their relative positions from the N-terminal, e.g., the left mostresidue, which is numbered 1, in a peptide sequence.

The term “mutant polypeptide” or “mutein” refers to a form of a peptidethat differs from its corresponding wild-type form or naturally existingform. A mutant peptide can contain one or more mutations, e.g.,replacement, insertion, deletion, etc. which result in the mutantpeptide.

The term “peptide conjugate,” refers to species of the invention inwhich a peptide is glycoconjugated with a modified sugar as set forthherein. In a representative example, the peptide is a mutant peptidehaving an O-linked glycosylation site not present in the wild-typepeptide.

“Proximate a proline residue,” as used herein refers to an amino acidthat is less than about 10 amino acids removed from a proline residue,preferably, less than about 9, 8, 7, 6 or 5 amino acids removed from aproline residue, more preferably, less than about 4, 3, 2 or 1 residuesremoved from a proline residue. The amino acid “proximate a prolineresidue” may be on the C- or N-terminal side of the proline residue.

The term “sialic acid” refers to any member of a family of nine-carboncarboxylated sugars. The most common member of the sialic acid family isN-acetyl-neuraminic acid(2-keto-5-acetamido-3,5-dideoxy-D-glycero-D-galactononulopyranos-1-onicacid (often abbreviated as Neu5Ac, NeuAc, or NANA). A second member ofthe family is N-glycolyl-neuraminic acid (Neu5Gc or NeuGc), in which theN-acetyl group of NeuAc is hydroxylated. A third sialic acid familymember is 2-keto-3-deoxy-nonulosonic acid (KDN) (Nadano et al. (1986) J.Biol. Chem. 261: 11550-11557; Kanamori et al., J. Biol. Chem. 265:21811-21819 (1990)). Also included are 9-substituted sialic acids suchas a 9-O—C₁-C₆ acyl-Neu5Ac like 9-O-lactyl-Neu5Ac or 9-O-acetyl-Neu5Ac,9-deoxy-9-fluoro-Neu5Ac and 9-azido-9-deoxy-Neu5Ac. For review of thesialic acid family, see, e.g., Varki, Glycobiology 2: 25-40 (1992);Sialic Acids Chemistry, Metabolism and Function, R. Schauer, Ed.(Springer-Verlag, New York (1992)). The synthesis and use of sialic acidcompounds in a sialylation procedure is disclosed in internationalapplication WO 92/16640, published Oct. 1, 1992.

As used herein, the term “modified sugar,” refers to a naturally- ornon-naturally-occurring carbohydrate that is enzymatically added onto anamino acid or a glycosyl residue of a peptide in a process of theinvention. The modified sugar is selected from a number of enzymesubstrates including, but not limited to sugar nucleotides (mono-, di-,and tri-phosphates), activated sugars (e.g., glycosyl halides, glycosylmesylates) and sugars that are neither activated nor nucleotides. The“modified sugar” is covalently functionalized with a “modifying group.”Useful modifying groups include, but are not limited to, water-solublepolymers, therapeutic moieties, diagnostic moieties, biomolecules andthe like. The modifying group is preferably not a naturally occurring,or an unmodified carbohydrate. The locus of functionalization with themodifying group is selected such that it does not prevent the “modifiedsugar” from being added enzymatically to a peptide.

The term “water-soluble” refers to moieties that have some detectabledegree of solubility in water. Methods to detect and/or quantify watersolubility are well known in the art. Exemplary water-soluble polymersinclude peptides, saccharides, poly(ethers), poly(amines),poly(carboxylic acids) and the like. Peptides can have mixed sequencesof be composed of a single amino acid, e.g., poly(lysine). An exemplarypolysaccharide is poly(sialic acid). An exemplary poly(ether) ispoly(ethylene glycol), e.g., m-PEG. Poly(ethylene imine) is an exemplarypolyamine, and poly(acrylic) acid is a representative poly(carboxylicacid).

The polymer backbone of the water-soluble polymer can be poly(ethyleneglycol) (i.e. PEG). However, it should be understood that other relatedpolymers are also suitable for use in the practice of this invention andthat the use of the term PEG or poly(ethylene glycol) is intended to beinclusive and not exclusive in this respect. The term PEG includespoly(ethylene glycol) in any of its forms, including alkoxy PEG,difunctional PEG, multiarmed PEG, forked PEG, branched PEG, pendent PEG(i.e. PEG or related polymers having one or more functional groupspendent to the polymer backbone), or PEG with degradable linkagestherein.

The polymer backbone can be linear or branched. Branched polymerbackbones are generally known in the art. Typically, a branched polymerhas a central branch core moiety and a plurality of linear polymerchains linked to the central branch core. PEG is commonly used inbranched forms that can be prepared by addition of ethylene oxide tovarious polyols, such as glycerol, pentaerythritol and sorbitol. Thecentral branch moiety can also be derived from several amino acids, suchas lysine. The branched poly(ethylene glycol) can be represented ingeneral form as R(—PEG-OH)_(m) in which R represents the core moiety,such as glycerol or pentaerythritol, and m represents the number ofarms. Multi-armed PEG molecules, such as those described in U.S. Pat.No. 5,932,462, which is incorporated by reference herein in itsentirety, can also be used as the polymer backbone.

Many other polymers are also suitable for the invention. Polymerbackbones that are non-peptidic and water-soluble, with from 2 to about300 termini, are particularly useful in the invention. Examples ofsuitable polymers include, but are not limited to, other poly(alkyleneglycols), such as poly(propylene glycol) (“PPG”), copolymers of ethyleneglycol and propylene glycol and the like, poly(oxyethylated polyol),poly(olefinic alcohol), poly(vinylpyrrolidone),poly(hydroxypropylmethacrylamide), poly(α-hydroxy acid), poly(vinylalcohol), polyphosphazene, polyoxazoline, poly(N-acryloylmorpholine),such as described in U.S. Pat. No. 5,629,384, which is incorporated byreference herein in its entirety, and copolymers, terpolymers, andmixtures thereof. Although the molecular weight of each chain of thepolymer backbone can vary, it is typically in the range of from about100 Da to about 100,000 Da, often from about 6,000 Da to about 80,000Da.

The term “glycoconjugation,” as used herein, refers to the enzymaticallymediated conjugation of a modified sugar species to an amino acid orglycosyl residue of a polypeptide, e.g., a mutant human growth hormoneof the present invention. A subgenus of “glycoconjugation” is“glycol-PEGylation,” in which the modifying group of the modified sugaris poly(ethylene glycol), and alkyl derivative (e.g., m-PEG) or reactivederivative (e.g., H₂N-PEG, HOOC-PEG) thereof.

The terms “large-scale” and “industrial-scale” are used interchangeablyand refer to a reaction cycle that produces at least about 250 mg,preferably at least about 500 mg, and more preferably at least about 1gram of glycoconjugate at the completion of a single reaction cycle.

The term, “glycosyl linking group,” as used herein refers to a glycosylresidue to which a modifying group (e.g., PEG moiety, therapeuticmoiety, biomolecule) is covalently attached; the glycosyl linking groupjoins the modifying group to the remainder of the conjugate. In themethods of the invention, the “glycosyl linking group” becomescovalently attached to a glycosylated or unglycosylated peptide, therebylinking the agent to an amino acid and/or glycosyl residue on thepeptide. A “glycosyl linking group” is generally derived from a“modified sugar” by the enzymatic attachment of the “modified sugar” toan amino acid and/or glycosyl residue of the peptide. The glycosyllinking group can be a saccharide-derived structure that is degradedduring formation of modifying group-modified sugar cassette (e.g.,oxidation→Schiff base formation→reduction), or the glycosyl linkinggroup may be intact. An “intact glycosyl linking group” refers to alinking group that is derived from a glycosyl moiety in which thesaccharide monomer that links the modifying group and to the remainderof the conjugate is not degraded, e.g., oxidized, e.g., by sodiummetaperiodate. “Intact glycosyl linking groups” of the invention may bederived from a naturally occurring oligosaccharide by addition ofglycosyl unit(s) or removal of one or more glycosyl unit from a parentsaccharide structure.

The term “targeting moiety,” as used herein, refers to species that willselectively localize in a particular tissue or region of the body. Thelocalization is mediated by specific recognition of moleculardeterminants, molecular size of the targeting agent or conjugate, ionicinteractions, hydrophobic interactions and the like. Other mechanisms oftargeting an agent to a particular tissue or region are known to thoseof skill in the art. Exemplary targeting moieties include antibodies,antibody fragments, transferrin, HS-glycoprotein, coagulation factors,serum proteins, β-glycoprotein, G-CSF, GM-CSF, M-CSF, EPO and the like.

As used herein, “therapeutic moiety” means any agent useful for therapyincluding, but not limited to, antibiotics, anti-inflammatory agents,anti-tumor drugs, cytotoxins, and radioactive agents. “Therapeuticmoiety” includes prodrugs of bioactive agents, constructs in which morethan one therapeutic moiety is bound to a carrier, e.g, multivalentagents. Therapeutic moiety also includes proteins and constructs thatinclude proteins. Exemplary proteins include, but are not limited to,Erythropoietin (EPO), Granulocyte Colony Stimulating Factor (GCSF),Granulocyte Macrophage Colony Stimulating Factor (GMCSF), Interferon(e.g., Interferon-α, -β, -γ), Interleukin (e.g., Interleukin II), serumproteins (e.g., Factors VII, VIa, VIII, IX, and X), Human ChorionicGonadotropin (HCG), Follicle Stimulating Hormone (FSH) and LutenizingHormone (LH) and antibody fusion proteins (e.g. Tumor Necrosis FactorReceptor ((TNFR)/Fc domain fusion protein)).

As used herein, “anti-tumor drug” means any agent useful to combatcancer including, but not limited to, cytotoxins and agents such asantimetabolites, alkylating agents, anthracyclines, antibiotics,antimitotic agents, procarbazine, hydroxyurea, asparaginase,corticosteroids, interferons and radioactive agents. Also encompassedwithin the scope of the term “anti-tumor drug,” are conjugates ofpeptides with anti-tumor activity, e.g. TNF-α. Conjugates include, butare not limited to those formed between a therapeutic protein and aglycoprotein of the invention. A representative conjugate is that formedbetween PSGL-1 and TNF-α.

As used herein, “a cytotoxin or cytotoxic agent” means any agent that isdetrimental to cells. Examples include taxol, cytochalasin B, gramicidinD, ethidium bromide, emetine, mitomycin, etoposide, tenoposide,vincristine, vinblastine, colchicin, doxorubicin, daunorubicin,dihydroxy anthracinedione, mitoxantrone, mithramycin, actinomycin D,1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine,propranolol, and puromycin and analogs or homologs thereof. Other toxinsinclude, for example, ricin, CC-1065 and analogues, the duocarmycins.Still other toxins include diptheria toxin, and snake venom (e.g., cobravenom).

As used herein, “a radioactive agent” includes any radioisotope that iseffective in diagnosing or destroying a tumor. Examples include, but arenot limited to, indium-111, cobalt-60. Additionally, naturally occurringradioactive elements such as uranium, radium, and thorium, whichtypically represent mixtures of radioisotopes, are suitable examples ofa radioactive agent. The metal ions are typically chelated with anorganic chelating moiety.

Many useful chelating groups, crown ethers, cryptands and the like areknown in the art and can be incorporated into the compounds of theinvention (e.g., EDTA, DTPA, DOTA, NTA, HDTA, etc. and their phosphonateanalogs such as DTPP, EDTP, HDTP, NTP, etc). See, for example, Pitt etal., “The Design of Chelating Agents for the Treatment of IronOverload,” In, INORGANIC CHEMISTRY IN BIOLOGY AND MEDICINE; Martell,Ed.; American Chemical Society, Washington, D.C., 1980, pp. 279-312;Lindoy, THE CHEMISTRY OF MACROCYCLIC LIGAND COMPLEXES; CambridgeUniversity Press, Cambridge, 1989; Dugas, BIOORGANIC CHEMISTRY;Springer-Verlag, New York, 1989, and references contained therein.

Additionally, a manifold of routes allowing the attachment of chelatingagents, crown ethers and cyclodextrins to other molecules is availableto those of skill in the art. See, for example, Meares et al.,“Properties of In Vivo Chelate-Tagged Proteins and Polypeptides.” In,MODIFICATION OF PROTEINS: FOOD, NUTRITIONAL, AND PHARMACOLOGICALASPECTS;” Feeney, et al., Eds., American Chemical Society, Washington,D.C., 1982, pp. 370-387; Kasina et al., Bioconjugate Chem., 9: 108-117(1998); Song et al., Bioconjugate Chem., 8: 249-255 (1997).

As used herein, “pharmaceutically acceptable carrier” includes anymaterial, which when combined with the conjugate retains the conjugates'activity and is non-reactive with the subject's immune systems. Examplesinclude, but are not limited to, any of the standard pharmaceuticalcarriers such as a phosphate buffered saline solution, water, emulsionssuch as oil/water emulsion, and various types of wetting agents. Othercarriers may also include sterile solutions, tablets including coatedtablets and capsules. Typically such carriers contain excipients such asstarch, milk, sugar, certain types of clay, gelatin, stearic acid orsalts thereof, magnesium or calcium stearate, talc, vegetable fats oroils, gums, glycols, or other known excipients. Such carriers may alsoinclude flavor and color additives or other ingredients. Compositionscomprising such carriers are formulated by well known conventionalmethods.

As used herein, “administering” means oral administration,administration as a suppository, topical contact, intravenous,intraperitoneal, intramuscular, intralesional, or subcutaneousadministration, administration by inhalation, or the implantation of aslow-release device, e.g., a mini-osmotic pump, to the subject.Administration is by any route including parenteral and transmucosal(e.g., oral, nasal, vaginal, rectal, or transdermal), particularly byinhalation. Parenteral administration includes, e.g., intravenous,intramuscular, intra-arteriole, intradermal, subcutaneous,intraperitoneal, intraventricular, and intracranial. Moreover, whereinjection is to treat a tumor, e.g., induce apoptosis, administrationmay be directly to the tumor and/or into tissues surrounding the tumor.Other modes of delivery include, but are not limited to, the use ofliposomal formulations, intravenous infusion, transdermal patches, etc.

The term “ameliorating” or “ameliorate” refers to any indicia of successin the treatment of a pathology or condition, including any objective orsubjective parameter such as abatement, remission or diminishing ofsymptoms or an improvement in a patient's physical or mental well-being.Amelioration of symptoms can be based on objective or subjectiveparameters; including the results of a physical examination and/or apsychiatric evaluation.

The term “therapy” refers to “treating” or “treatment” of a disease orcondition including preventing the disease or condition from occurringin an animal that may be predisposed to the disease but does not yetexperience or exhibit symptoms of the disease (prophylactic treatment),inhibiting the disease (slowing or arresting its development), providingrelief from the symptoms or side-effects of the disease (includingpalliative treatment), and relieving the disease (causing regression ofthe disease).

The term “effective amount” or “an amount effective to” or a“therapeutically effective amount” or any grammatically equivalent termmeans the amount that, when administered to an animal for treating adisease, is sufficient to effect treatment for that disease.

The term “isolated” refers to a material that is substantially oressentially free from components, which are used to produce thematerial. For peptide conjugates of the invention, the term “isolated”refers to material that is substantially or essentially free fromcomponents, which normally accompany the material in the mixture used toprepare the peptide conjugate. “Isolated” and “pure” are usedinterchangeably. Typically, isolated peptide conjugates of the inventionhave a level of purity preferably expressed as a range. The lower end ofthe range of purity for the peptide conjugates is about 60%, about 70%or about 80% and the upper end of the range of purity is about 70%,about 80%, about 90% or more than about 90%.

When the peptide conjugates are more than about 90% pure, their puritiesare also preferably expressed as a range. The lower end of the range ofpurity is about 90%, about 92%, about 94%, about 96% or about 98%. Theupper end of the range of purity is about 92%, about 94%, about 96%,about 98% or about 100% purity.

Purity is determined by any art-recognized method of analysis (e.g.,band intensity on a silver stained gel, polyacrylamide gelelectrophoresis, HPLC, or a similar means).

“Essentially each member of the population,” as used herein, describes acharacteristic of a population of peptide conjugates of the invention inwhich a selected percentage of the modified sugars added to a peptideare added to multiple, identical acceptor sites on the peptide.“Essentially each member of the population” speaks to the “homogeneity”of the sites on the peptide conjugated to a modified sugar and refers toconjugates of the invention, which are at least about 80%, preferably atleast about 90% and more preferably at least about 95% homogenous.

“Homogeneity,” refers to the structural consistency across a populationof acceptor moieties to which the modified sugars are conjugated. Thus,in a peptide conjugate of the invention in which each modified sugarmoiety is conjugated to an acceptor site having the same structure asthe acceptor site to which every other modified sugar is conjugated, thepeptide conjugate is said to be about 100% homogeneous. Homogeneity istypically expressed as a range. The lower end of the range ofhomogeneity for the peptide conjugates is about 60%, about 70% or about80% and the upper end of the range of purity is about 70%, about 80%,about 90% or more than about 90%.

When the peptide conjugates are more than or equal to about 90%homogeneous, their homogeneity is also preferably expressed as a range.The lower end of the range of homogeneity is about 90%, about 92%, about94%, about 96% or about 98%. The upper end of the range of purity isabout 92%, about 94%, about 96%, about 98% or about 100% homogeneity.The purity of the peptide conjugates is typically determined by one ormore methods known to those of skill in the art, e.g., liquidchromatography-mass spectrometry (LC-MS), matrix assisted laserdesorption mass time of flight spectrometry (MALDITOF), capillaryelectrophoresis, and the like.

“Substantially uniform glycoform” or a “substantially uniformglycosylation pattern,” when referring to a glycopeptide species, refersto the percentage of acceptor moieties that are glycosylated by theglycosyltransferase of interest (e.g., fucosyltransferase). For example,in the case of a α1,2 fucosyltransferase, a substantially uniformfucosylation pattern exists if substantially all (as defined below) ofthe Galβ1,4-GlcNAc-R and sialylated analogues thereof are fucosylated ina peptide conjugate of the invention. It will be understood by one ofskill in the art, that the starting material may contain glycosylatedacceptor moieties (e.g., fucosylated Galβ1,4-GlcNAc-R moieties). Thus,the calculated percent glycosylation will include acceptor moieties thatare glycosylated by the methods of the invention, as well as thoseacceptor moieties already glycosylated in the starting material.

The term “substantially” in the above definitions of “substantiallyuniform” generally means at least about 40%, at least about 70%, atleast about 80%, or more preferably at least about 90%, and still morepreferably at least about 95% of the acceptor moieties for a particularglycosyltransferase are glycosylated.

Where substituent groups are specified by their conventional chemicalformulae, written from left to right, they equally encompass thechemically identical substituents, which would result from writing thestructure from right to left, e.g., —CH₂O— is intended to also recite—OCH₂—.

The term “alkyl,” by itself or as part of another substituent means,unless otherwise stated, a straight or branched chain, or cyclichydrocarbon radical, or combination thereof, which may be fullysaturated, mono- or polyunsaturated and can include di- and multivalentradicals, having the number of carbon atoms designated (i.e. C₁-C₁₀means one to ten carbons). Examples of saturated hydrocarbon radicalsinclude, but are not limited to, groups such as methyl, ethyl, n-propyl,isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl,(cyclohexyl)methyl, cyclopropylmethyl, homologs and isomers of, forexample, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. Anunsaturated alkyl group is one having one or more double bonds or triplebonds. Examples of unsaturated alkyl groups include, but are not limitedto, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl),2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl,3-butynyl, and the higher homologs and isomers. The term “alkyl,” unlessotherwise noted, is also meant to include those derivatives of alkyldefined in more detail below, such as “heteroalkyl.” Alkyl groups thatare limited to hydrocarbon groups are termed “homoalkyl”.

The term “alkylene” by itself or as part of another substituent means adivalent radical derived from an alkane, as exemplified, but notlimited, by —CH₂CH₂CH₂CH₂—, and further includes those groups describedbelow as “heteroalkylene.” Typically, an alkyl (or alkylene) group willhave from 1 to 24 carbon atoms, with those groups having 10 or fewercarbon atoms being preferred in the present invention. A “lower alkyl”or “lower alkylene” is a shorter chain alkyl or alkylene group,generally having eight or fewer carbon atoms.

The terms “alkoxy,” “alkylamino” and “alkylthio” (or thioalkoxy) areused in their conventional sense, and refer to those alkyl groupsattached to the remainder of the molecule via an oxygen atom, an aminogroup, or a sulfur atom, respectively.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a stable straight or branched chain, orcyclic hydrocarbon radical, or combinations thereof, consisting of thestated number of carbon atoms and at least one heteroatom selected fromthe group consisting of O, N, Si and S, and wherein the nitrogen andsulfur atoms may optionally be oxidized and the nitrogen heteroatom mayoptionally be quaternized. The heteroatom(s) O, N and S and Si may beplaced at any interior position of the heteroalkyl group or at theposition at which the alkyl group is attached to the remainder of themolecule. Examples include, but are not limited to, —CH₂—CH₂—O—CH₃,—CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃, —CH₂—S—CH₂—CH₃, —CH₂—CH₂,—S(O)—CH₃, —CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃, —Si(CH₃)₃, —CH₂—CH═N—OCH₃,and —CH═CH—N(CH₃)—CH₃. Up to two heteroatoms may be consecutive, suchas, for example, —CH₂—NH—OCH₃ and —CH₂—O—Si(CH₃)₃. Similarly, the term“heteroalkylene” by itself or as part of another substituent means adivalent radical derived from heteroalkyl, as exemplified, but notlimited by, —CH₂—CH₂—S—CH₂—CH₂— and —CH₂—S—CH₂—CH₂—NH—CH₂—. Forheteroalkylene groups, heteroatoms can also occupy either or both of thechain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino,alkylenediamino, and the like). Still further, for alkylene andheteroalkylene linking groups, no orientation of the linking group isimplied by the direction in which the formula of the linking group iswritten. For example, the formula —C(O)₂R′— represents both —C(O)₂R′-and —R′C(O)₂—.

The terms “cycloalkyl” and “heterocycloalkyl”, by themselves or incombination with other terms, represent, unless otherwise stated, cyclicversions of “alkyl” and “heteroalkyl”, respectively. Additionally, forheterocycloalkyl, a heteroatom can occupy the position at which theheterocycle is attached to the remainder of the molecule. Examples ofcycloalkyl include, but are not limited to, cyclopentyl, cyclohexyl,1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples ofheterocycloalkyl include, but are not limited to,1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl,3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,1-piperazinyl, 2-piperazinyl, and the like.

The terms “halo” or “halogen,” by themselves or as part of anothersubstituent, mean, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom. Additionally, terms such as “haloalkyl,” aremeant to include monohaloalkyl and polyhaloalkyl. For example, the term“halo(C₁-C₄)alkyl” is mean to include, but not be limited to,trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, andthe like.

The term “aryl” means, unless otherwise stated, a polyunsaturated,aromatic, substituent that can be a single ring or multiple rings(preferably from 1 to 3 rings), which are fused together or linkedcovalently. The term “heteroaryl” refers to aryl groups (or rings) thatcontain from one to four heteroatoms selected from N, O, and S, whereinthe nitrogen and sulfur atoms are optionally oxidized, and the nitrogenatom(s) are optionally quaternized. A heteroaryl group can be attachedto the remainder of the molecule through a heteroatom. Non-limitingexamples of aryl and heteroaryl groups include phenyl, 1-naphthyl,2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl,2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl,2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl,5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl,2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl,4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl,1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl,3-quinolyl, tetrazolyl, benzo[b]furanyl, benzo[b]thienyl,2,3-dihydrobenzo[1,4]dioxin-6-yl, benzo[1,3]dioxol-5-yl and 6-quinolyl.Substituents for each of the above noted aryl and heteroaryl ringsystems are selected from the group of acceptable substituents describedbelow.

For brevity, the term “aryl” when used in combination with other terms(e.g., aryloxy, arylthioxy, arylalkyl) includes both aryl and heteroarylrings as defined above. Thus, the term “arylalkyl” is meant to includethose radicals in which an aryl group is attached to an alkyl group(e.g., benzyl, phenethyl, pyridylmethyl and the like) including thosealkyl groups in which a carbon atom (e.g., a methylene group) has beenreplaced by, for example, an oxygen atom (e.g., phenoxymethyl,2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl, and the like).

Each of the above terms (e.g., “alkyl,” “heteroalkyl,” “aryl” and“heteroaryl”) is meant to include both substituted and unsubstitutedforms of the indicated radical. Preferred substituents for each type ofradical are provided below.

Substituents for the alkyl and heteroalkyl radicals (including thosegroups often referred to as alkylene, alkenyl, heteroalkylene,heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) are generically referred to as “alkyl groupsubstituents,” and they can be one or more of a variety of groupsselected from, but not limited to: —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′,-halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″ R′″)═NR′″,—NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN and—NO₂ in a number ranging from zero to (2m′+1), where m′ is the totalnumber of carbon atoms in such radical. R′, R″, R′″ and R″″ eachpreferably independently refer to hydrogen, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted aryl, e.g., aryl substitutedwith 1-3 halogens, substituted or unsubstituted alkyl, alkoxy orthioalkoxy groups, or arylalkyl groups. When a compound of the inventionincludes more than one R group, for example, each of the R groups isindependently selected as are each R′, R″, R′″ and R″″ groups when morethan one of these groups is present. When R′ and R″ are attached to thesame nitrogen atom, they can be combined with the nitrogen atom to forma 5-, 6-, or 7-membered ring. For example, —NR′R″ is meant to include,but not be limited to, 1-pyrrolidinyl and 4-morpholinyl. From the abovediscussion of substituents, one of skill in the art will understand thatthe term “alkyl” is meant to include groups including carbon atoms boundto groups other than hydrogen groups, such as haloalkyl (e.g., —CF₃ and—CH₂CF₃) and acyl (e.g., —C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and thelike).

Similar to the substituents described for the alkyl radical,substituents for the aryl and heteroaryl groups are generically referredto as “aryl group substituents.” The substituents are selected from, forexample: halogen, —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′, -halogen,—SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR″″,—NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN and—NO₂, —R′, —N₃, —CH(Ph)₂, fluoro(C₁-C₄)alkoxy, and fluoro(C₁-C₄)alkyl,in a number ranging from zero to the total number of open valences onthe aromatic ring system; and where R′, R″, R′″ and R″″ are preferablyindependently selected from hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted aryl and substituted or unsubstituted heteroaryl. When acompound of the invention includes more than one R group, for example,each of the R groups is independently selected as are each R′, R″, R′″and R″″ groups when more than one of these groups is present. In theschemes that follow, the symbol X represents “R” as described above.

Two of the substituents on adjacent atoms of the aryl or heteroaryl ringmay optionally be replaced with a substituent of the formula-T-C(O)—(CRR′)_(q)—U—, wherein T and U are independently —NR—, —O—,—CRR′- or a single bond, and q is an integer of from 0 to 3.Alternatively, two of the substituents on adjacent atoms of the aryl orheteroaryl ring may optionally be replaced with a substituent of theformula -A-(CH₂)_(r)—B—, wherein A and B are independently —CRR′—, —O—,—NR—, —S—, —S(O)—, —S(O)₂—, —S(O)₂NR′- or a single bond, and r is aninteger of from 1 to 4. One of the single bonds of the new ring soformed may optionally be replaced with a double bond. Alternatively, twoof the substituents on adjacent atoms of the aryl or heteroaryl ring mayoptionally be replaced with a substituent of the formula—(CRR′)_(s)—X—(CR″R′″)_(d)—, where s and d are independently integers offrom 0 to 3, and X is —O—, —NR′—, —S—, —S(O)—, —S(O)₂—, or —S(O)₂NR′—.The substituents R, R′, R″ and R′″ are preferably independently selectedfrom hydrogen or substituted or unsubstituted (C₁-C₆)alkyl.

As used herein, the term “heteroatom” is meant to include oxygen (O),nitrogen (N), sulfur (S) and silicon (Si).

Introduction

The present invention provides conjugates of glycopeptides in which amodified sugar moiety is attached either directly or indirectly (e.g.,through and intervening glycosyl residue) to an O-linked glycosylationsite on the peptide. Also provided are methods for producing theconjugates of the invention.

The O-linked glycosylation site is generally the hydroxy side chain of anatural (e.g., serine, threonine) or unnatural (e.g., 5-hydroxyprolineor 5-hydroxylysine) amino acid. Exemplary O-linked saccharyl residuesinclude N-acetylgalactosamine, galactose, mannose, GlcNAc, glucose,fucose or xylose.

The methods of the invention can be practiced on any peptide having anO-linked glycosylation site. For example, the methods are of use toproduce O-linked glycoconjugates in which the glycosyl moiety isattached to an O-linked glycosylation site that is present in the wildtype peptide. Accordingly, the present invention providesglycoconjugates of wild-type peptides that include an O-linkedglycosylation site. Exemplary peptides according to this descriptioninclude G-CSF, GM-CSF, IL-2 and interferon.

In exemplary embodiments the invention also provides novel mutantpeptides that include one or more O-linked glycosylation sites that arenot present in the corresponding wild-type peptide. In one embodimentthe mutant polypeptide is a G-CSF polypeptide. In other exemplaryembodiments the mutant polypeptide is an hGH polypeptide, an IFN alphapolypeptide or a GM-CSF polypeptide. Also provided are O-linkedglycosylated versions of the mutant peptides, and methods of preparingO-linked glycosylated mutant peptides. Additional methods include theelaboration, trimming back and/or modification of the O-linked glycosylresidue and glycosyl residues that are N—, rather than O-linked.

In an exemplary aspect, the invention provides a mutant peptide havingthe formula:

in which AA is an amino acid with a side chain that includes a hydroxylmoiety. Exemplary hydroxyamino acids are threonine and serine. TheGalNAc moiety is linked to AA through the oxygen atom of the hydroxylmoiety. AA may be present in the wild type peptide or, alternatively, itis added or relocated by mutating the sequence of the wild type peptide.X is a modifying group, a saccharyl moiety, e.g., sialyl, galactosyl andGal-Sia groups, or a saccharyl moiety and a modifying group. In anexemplary embodiment, in which X is a saccharyl moiety, it includes amodifying group, as discussed herein. The glycosylated amino acid can beat the N- or C-peptide terminus or internal to the peptide sequence.

In an exemplary embodiment, X comprises a group selected from sialyl,galactosyl and Gal-Sia moieties, wherein at least one of said sialyl,galactosyl and Gal-Sia comprises a modifying group. In a furtherexemplary embodiment X comprises the moiety:

wherein D is a member selected from —OH and R¹-L-HN—; G is a memberselected from R¹-L- and —C(O)(C₁-C₆)alkyl; R¹ is a moiety comprising amember selected a moiety comprising a straight-chain or branchedpoly(ethylene glycol) residue; and L is a linker which is a memberselected from a bond, substituted or unsubstituted alkyl and substitutedor unsubstituted heteroalkyl, such that when D is OH, G is R¹-L-, andwhen G is —C(O)(C₁-C₆)alkyl, D is R¹-L-NH—.

In another exemplary embodiment X comprises the structure:

in which L is a substituted or unsubstituted alkyl or substituted orunsubstituted heteroalkyl group; and n is selected from the integersfrom 0 to about 2500. In yet another exemplary embodiment X comprisesthe structure:

in which s is selected from the integers from 0 to 20.

In another exemplary embodiment, AA is located within a proline-richsegment of the mutant peptide and/or it is proximate to a prolineresidue. Appropriate sequences forming O-linked glycosylation sites arereadily determined by interrogating the enzymatic O-linked glycosylationof short peptides containing one or more putative O-linked glycosylationsites.

The conjugates of the invention are formed between peptides and diversespecies such as water-soluble polymers, therapeutic moieties, diagnosticmoieties, targeting moieties and the like. Also provided are conjugatesthat include two or more peptides linked together through a linker arm,i.e., multifunctional conjugates; at least one peptide beingO-glycosylated or including a mutant O-linked glycosylation site. Themulti-functional conjugates of the invention can include two or morecopies of the same peptide or a collection of diverse peptides withdifferent structures, and/or properties. In exemplary conjugatesaccording to this embodiment, the linker between the two peptides isattached to at least one of the peptides through an O-linked glycosylresidue, such as an O-linked glycosyl intact glycosyl linking group.

The conjugates of the invention are formed by the enzymatic attachmentof a modified sugar to the glycosylated or unglycosylated peptide. Themodified sugar is directly added to an O-linked glycosylation site, orto a glycosyl residue attached either directly or indirectly (e.g.,through one or more glycosyl residue) to an O-linked glycosylation site.The invention also provides a conjugate of an O-linked glycosylatedpeptide in which a modified sugar is directly attached to an N-linkedsite, or to a glycosyl residue attached either directly or indirectly toan N-linked glycosylation site.

The modified sugar, when interposed between the peptide (or glycosylresidue) and the modifying group on the sugar becomes what is referredto herein as “an intact glycosyl linking group.” Using the exquisiteselectivity of enzymes, such as glycosyltransferases, the present methodprovides peptides that bear a desired group at one or more specificlocations. Thus, according to the present invention, a modified sugar isattached directly to a selected locus on the peptide chain or,alternatively, the modified sugar is appended onto a carbohydrate moietyof a glycopeptide. Peptides in which modified sugars are bound to both aglycopeptide carbohydrate and directly to an amino acid residue of thepeptide backbone are also within the scope of the present invention.

In contrast to known chemical and enzymatic peptide elaborationstrategies, the methods of the invention, make it possible to assemblepeptides and glycopeptides that have a substantially homogeneousderivatization pattern; the enzymes used in the invention are generallyselective for a particular amino acid residue or combination of aminoacid residues of the peptide. The methods are also practical forlarge-scale production of modified peptides and glycopeptides. Thus, themethods of the invention provide a practical means for large-scalepreparation of glycopeptides having preselected uniform derivatizationpatterns. The methods are particularly well suited for modification oftherapeutic peptides, including but not limited to, glycopeptides thatare incompletely glycosylated during production in cell culture cells(e.g., mammalian cells, insect cells, plant cells, fungal cells, yeastcells, or prokaryotic cells) or transgenic plants or animals.

The methods of the invention also provide conjugates of glycosylated andunglycosylated peptides with increased therapeutic half-life due to, forexample, reduced clearance rate, or reduced rate of uptake by the immuneor reticuloendothelial system (RES). Moreover, the methods of theinvention provide a means for masking antigenic determinants onpeptides, thus reducing or eliminating a host immune response againstthe peptide. Selective attachment of targeting agents to a peptide usingan appropriate modified sugar can also be used to target a peptide to aparticular tissue or cell surface receptor that is specific for theparticular targeting agent. Moreover, there is provided a class ofpeptides that are specifically modified with a therapeutic moietyconjugated through a glycosyl linking group.

O-Glycosylation

The present invention provides O-linked glycosylated peptides,conjugates of these species and methods for forming O-linkedglycosylated peptides that include a selected amino acid sequence (“anO-linked glycosylation site”). Of particular interest are mutantpeptides that include an O-linked glycosylation site that is not presentin the corresponding wild type peptide. The O-linked glycosylation siteis a locus for attachment of a glycosyl residue that bears a modifyinggroup.

Mucin-type O-linked glycosylation, one of the most abundant forms ofprotein glycosylation, is found on secreted and cell surface associatedglycoproteins of all eukaryotic cells. There is great diversity in thestructures created by O-linked glycosylation (hundreds of potentialstructures), which are produced by the catalytic activity of hundreds ofglycosyltransferase enzymes that are resident in the Golgi complex.Diversity exists at the level of the glycan structure and in positionsof attachment of O-glycans to protein backbones. Despite the high degreeof potential diversity, it is clear that O-linked glycosylation is ahighly regulated process that shows a high degree of conservation amongmulticellular organisms.

The first step in mucin-type O-linked glycosylation is catalysed by oneor more members of a large family of UDP-GalNAc: polypeptideN-acetylgalactosaminyltransferases (GalNAc-transferases) (EC 2.4.1.41),which transfer GalNAc to serine and threonine acceptor sites (Hassan etal., J. Biol. Chem. 275: 38197-38205 (2000)). To date twelve members ofthe mammalian GalNAc-transferase family have been identified andcharacterized (Schwientek et al., J. Biol. Chem. 277: 22623-22638(2002)), and several additional putative members of this gene familyhave been predicted from analysis of genome databases. TheGalNAc-transferase isoforms have different kinetic properties and showdifferential expression patterns temporally and spatially, suggestingthat they have distinct biological functions (Hassan et al., J. Biol.Chem. 275: 38197-38205 (2000)). Sequence analysis of GalNAc-transferaseshave led to the hypothesis that these enzymes contain two distinctsubunits: a central catalytic unit, and a C-terminal unit with sequencesimilarity to the plant lectin ricin, designated the “lectin domain”(Hagen et al., J. Biol. Chem. 274: 6797-6803 (1999); Hazes, Protein Eng.10: 1353-1356 (1997); Breton et al., Curr. Opin. Struct. Biol. 9:563-571 (1999)). Previous experiments involving site-specificmutagenesis of selected conserved residues confirmed that mutations inthe catalytic domain eliminated catalytic activity. In contrast,mutations in the “lectin domain” had no significant effects on catalyticactivity of the GalNAc-transferase isoform, GalNAc-T1 (Tenno et al., J.Biol. Chem. 277(49): 47088-96 (2002)). Thus, the C-terminal “lectindomain” was believed not to be functional and not to play roles for theenzymatic functions of GalNAc-transferases (Hagen et al., J. Biol. Chem.274: 6797-6803 (1999)).

However, recent evidence demonstrates that some GalNAc-transferasesexhibit unique activities with partially GalNAc-glycosylatedglycopeptides. The catalytic actions of at least threeGalNAc-transferase isoforms, GalNAc-T4, -T7, and -T10, selectively acton glycopeptides corresponding to mucin tandem repeat domains where onlysome of the clustered potential glycosylation sites have been GalNAcglycosylated by other GalNAc-transferases (Bennett et al., FEBS Letters460: 226-230 (1999); Ten Hagen et al., J. Biol. Chem. 276: 17395-17404(2001); Bennett et al., J. Biol. Chem. 273: 30472-30481 (1998); TenHagen et al., J. Biol. Chem. 274: 27867-27874 (1999)). GalNAc-T4 and -T7recognize different GalNAc-glycosylated peptides and catalyse transferof GalNAc to acceptor substrate sites in addition to those that werepreviously utilized. One of the functions of such GalNAc-transferaseactivities is predicted to represent a control step of the density ofO-glycan occupancy in mucins and mucin-like glycoproteins with highdensity of O-linked glycosylation.

One example of this is the glycosylation of the cancer-associated mucinMUC1. MUC1 contains a tandem repeat O-linked glycosylated region of 20residues (HGVTSAPDTRPAPGSTAPPA) with five potential O-linkedglycosylation sites. GalNAc-T1, -T2, and -T3 can initiate glycosylationof the MUC1 tandem repeat and incorporate at only three sites(HGVTSAPDTRPAPGSTAPPA, GalNAc attachment sites underlined). GalNAc-T4 isunique in that it is the only GalNAc-transferase isoform identified sofar that can complete the O-linked glycan attachment to all fiveacceptor sites in the 20 amino acid tandem repeat sequence of the breastcancer associated mucin, MUC1. GalNAc-T4 transfers GalNAc to at leasttwo sites not used by other GalNAc-transferase isoforms on theGalNAc₄TAP24 glycopeptide (TAPPAHGVTSAPDTRPAPGSTAPP, unique GalNAc-T4attachment sites are in bold) (Bennett et al., J. Biol. Chem. 273:30472-30481 (1998). An activity such as that exhibited by GalNAc-T4appears to be required for production of the glycoform of MUC1 expressedby cancer cells where all potential sites are glycosylated (Muller etal., J. Biol. Chem. 274: 18165-18172 (1999)). Normal MUC1 from lactatingmammary glands has approximately 2.6 O-linked glycans per repeat (Mulleret al., J. Biol. Chem. 272: 24780-24793 (1997) and MUC1 derived from thecancer cell line T47D has 4.8 O-linked glycans per repeat (Muller etal., J. Biol. Chem. 274: 18165-18172 (1999)). The cancer-associated formof MUC1 is therefore associated with higher density of O-linked glycanoccupancy and this is accomplished by a GalNAc-transferase activityidentical to or similar to that of GalNAc-T4.

Polypeptide GalNAc-transferases, which have not displayed apparentGalNAc-glycopeptide specificities, also appear to be modulated by theirputative lectin domains (PCT WO 01/85215 A2). Recently, it was foundthat mutations in the GalNAc-T1 putative lectin domain, similarly tothose previously analysed in GalNAc-T4 (Hassan et al., J. Biol. Chem.275: 38197-38205 (2000)), modified the activity of the enzyme in asimilar fashion as GalNAc-T4. Thus, while wild type GalNAc-T1 addedmultiple consecutive GalNAc residues to a peptide substrate withmultiple acceptor sites, mutated GalNAc-T1 failed to add more than oneGalNAc residue to the same substrate (Tenno et al, J. Biol. Chem.277(49): 47088-96 (2002)).

Since it has been demonstrated that mutations of GalNAc transferases canbe utilized to produce glycosylation patterns that are distinct fromthose produced by the wild-type enzymes, it is within the scope of thepresent invention to utilize one or more mutant GalNAc transferase inpreparing the O-linked glycosylated peptides of the invention.

Mutant Peptides with O-linked Glycosylation Sites

The peptides provided by the present invention include an amino acidsequence that is recognized as a GalNAc acceptor by one or morewild-type or mutant GalNac transferases. The amino acid sequence of thepeptide is either the wild-type, for those peptides that include anO-linked glycosylation site, a mutant sequence in which a non-naturallyocurring O-linked glycosylation site is introduced, or a polypeptidecomprising both naturally occurring and non-naturally occurring O-linkedglycosylation sites. Exemplary peptides with which the present inventionis practiced include granulocyte colony stimulating factor (G-CSF),e.g., 175 and 178 amino acid wild types (with or without N-terminalmethionine residues), interferon (e.g., interferon alpha, e.g.,interferon alpha 2b, or interferon alpha 2a), granulocyte macrophagecolony stimulating factor (GM-CSF), human growth hormone and interleukin(e.g., interleukin 2). The emphasis of the following discussion onG-CSF, GM-CSF and IFN-α2β is for clarity of illustration. Any number inthe superscript of an amino acid indicates the amino acid positionrelative to the N-terminal methionine of the polypeptide. These numberscan be readily adjusted to reflect the absence of N-terminal methionineif the N-terminal of the polypeptide starts without a methionine. It isunderstood that the N-terminals of the exemplary peptides can start withor without a methionine. In addition, those of skill will understandthat the strategy set forth herein for preparing O-linkedglycoconjugated analogues of wild-type and mutant peptides is applicableto any peptide.

In an exemplary embodiment, the peptide is a biologically active G-CSFmutant that includes one or more mutation at a site selected from theN-terminus, adjacent to or encompassing H⁵³, P⁶¹, P¹²⁹, P¹³³ and P¹⁷⁵.Biologically active G-CSF mutants of the present invention include anyG-CSF polypeptide, in part or in whole, with one or more mutations thatdo not result in substantial or entire loss of its biological activityas it is measured by any suitable functional assays known to one skilledin the art. In one embodiment, mutations within the biologically activeG-CSF mutants of the present invention are located within one or moreO-linked glycosylation sites that do not naturally exist in wild typeG-CSF. In another embodiment, mutations within the biologically activeG-CSF mutants of the present invention reside within as well as outsideof one or more O-linked glycosylation sites of the G-CSF mutants.

Representative wild type and mutant G-CSF polypeptides have sequencesthat are selected from:

SEQ. ID NO. 1 (178 amino acid wild type) mtplgpasslp qsfllkcleqvrkiqgdgaa lqeklvseca tyklchpeel vllghslgip waplsscpsq alqlagclsqlhsglflyqg llqalegisp elgptldtlq ldvadfatti wqqmeelgma palqptqgampafasafqrr aggvlvashl qsflevsyrv lrhlaqp; SEQ. ID NO. 2 (178 amino acidwild type without N-terminal methionine) tplgpasslp qsfllkcleqvrkiqgdgaa lqeklvseca tyklchpeel vllghslgip waplsscpsq alqlagclsqlhsglflyqg llqalegisp elgptldtlq ldvadfatti wqqmeelgma palqptqgampafasafqrr aggvlvashl qsflevsyrv lrhlaqp; SEQ. ID NO. 3 (175 amino acidwild type) mtplgpasslp qsfllkcleq vrkiqgdgaa lqeklca tyklchpeelvllghslgip waplsscpsq alqlagclsq lhsglflyqg llqalegip elgptldtlqldvadfatti wqqmeelgma palqptqgam pafasafqrr aggvlvashl qsflevsyrvlrhlaqp; SEQ. ID NO. 4 (175 amino acid wild type without N-terminalmethionine) mtplgpasslp qsfllkcleq vrkiqgdgaa lqeklca tyklchpeelvllghslgip waplsscpsq alqlagclsq lhsglflyqg llqalegisp elgptldtlqldvadfatti wqqmeelgma palqptqgam pafasafqrr aggvlvashl qsflevsyrvlrhlaqp; SEQ. ID NO. 5 mvtplgpasslp qsfllkcleq vrkiqgdgaa lqeklcatyklchpeel vllghslgip waplsscpsq alqlagclsq lhsglflyqg llqalegispelgptldtlq ldvadfatti wqqmeelgma palqptqgam pafasafqrr aggvlvashlqsflevsyrv lrhlaqp; SEQ. ID NO. 6 mvtplgpasslp qsfllkcleq vrkiqgdgaalqeklca tyklchpeel vllghtlgip waplsscpsq alqlagclsq lhsglflyqgllqalegisp elgptldtlq ldvadfatti wqqmeelgma palqptqgam pafasafqrraggvlvashl qsflevsyrv lrhlaqp; SEQ. ID NO. 7 mtplgpasslp qsfllkcleqvrkiqgdgaa lqeklca tyklchpeel vllghtlgip waplsscpsq alqlagclsqlhsglflyqg llqalegisp elgptldtlq ldvadfatti wqqmeelgma palqptqgampafasafqrr aggvlvashl qsflevsyrv lrhlaqp; SEQ. ID NO. 8 mvtplgpasslpqsfllkcleq vrkiqgdgaa lqeklca tyklchpeel vllgsslgip waplsscpsqalqlagclsq lhsglflyqg llqalegisp elgptldtlq ldvadfatti wqqmeelgmapalqptqgam pafasafqrr aggvlvashl qsflevsyrv lrhlaqp; SEQ. ID NO. 9mqtplgpasslp qsfllkcleq vrkiqgdgaa lqeklca tyklchpeel vllghslgipwaplsscpsq alqlagclsq lhsglflyqg llqalegisp elgptldtlq ldvadfattiwqqmeelgma palqptqgam pafasafqrr aggvlvashl qsflevsyrv lrhlaqp; SEQ. IDNO. 10 mtplgpasslp qsfllkcleq vrkiqgdgaa lqeklcaa tyklchpeel vllghslgipwaplsscpsq alqlagclsq lhsglflyqg llqalegisp elgptldtlq ldvadfattiwqqmeelgma palqptqgam pafasafqrr aggvlvashl qsflevsyrv lrhlaqptqgamp;and SEQ. ID NO. 11 mtplgpasslp qsfllkcleq vrkiqgdgaa lqeklcaa tyklchpeelvllgsslgip waplsscpsq alqlagclsq lhsglflyqg llqalegisp elgptldtlqldvadfatti wqqmeelgma palqptqgam pafasafqrr aggvlvashl qsflevsyrvlrhlaqp SEQ ID NO:12 maitplgpasslp qsfllkcleq vrkiqgdgaa lqeklcaatyklchpeelvll ghslgipwap lsscpsqalq lagclsqlhs glflyqgllq alegispeigptldtlqldv adfattiwqq meelgmapal qptqgampaf asafqrragg vlvashlqsflevsyrvlrh laqp SEQ ID NO:13 mgvtetplgpasslp qsfllkcleq vrkiqgdgaalqeklcaatyk lchpeelvll ghslgipwap lsscpsqalq lagclsqlhs glflyqgllqalegispeig ptldtlqldv adfattiwqq meelgmapal qptqgampaf asafqrraggvlvashlqsf levsyrvlrh laqp SEQ ID NO:14 maptplgpasslp qsfllkcleqvrkiqgdgaa lqeklcaatyk lchpeelvll ghslgipwap lsscpsqalq lagclsqlhsglflyqgllq alegispeig ptldtlqldv adfattiwqq meelgmapal qptqgampafasafqrragg vlvashlqsf levsyrvlrh laqp SEQ ID NO:15 Mtptqglgpasslpqsfllkcleq vrkiqgdgaa lqeklcaatyk lchpeelvll ghslgipwap lsscpsqalqlagclsqlhs glflyqgllq alegispeig ptldtlqldv adfattiwqq meelgmapalqptqgampaf asafqrragg vlvashlqsf levsyrvlrh laqp SEQ ID NO:16mtplgpasslp qsfllkcleq vrkiqgdgaa lqeklcaatyk lchpeelvll ghslgipwaplsscpsqalq lagclsqlhs glflyqgllq alegispeig ptldtlqldv adfattiwqqmeelgmapatqptqgampaf asafqrragg vlvashlqsf levsyrvlrh laqp SEQ ID NO:17Mtplgpasslp qsfllkcleq vrkiqgdgaa lqeklcaatyk lchpeelvll ghslgipftplsscpsqalq lagclsqlhs glflyqgllq alegispeig ptldtlqldv adfattiwqqmeelgmapaL qptqgampaf asafqrragg vlvashlqsf levsyrvlrh laqp SEQ ID NO:18mtplgpasslpqsfllkcleqvrkiqgdgaalqeklcatyklchpeelvllghslgipwaplsscpsqalqlagclsqlhsglflyqgllqalegispelgptldtlqldvadfattiwqqmeelgmapalqptq t ampafasafqrraggvlvashlqsflevsyrvlrhlaqp.

In another exemplary embodiment, the peptide is a biologically activehGH mutant that includes one or more mutations at a site selected fromthe N-terminus or adjacent to or encompassing P¹³³. Biologically activehGH mutants of the present invention include any hGH polypeptide, inpart or in whole, with one or more mutations that do not result insubstantial or entire loss of its biological activity as it is measuredby any suitable functional assays known to one skilled in the art. Inone embodiment, mutations within the biologically active hGH mutants ofthe present invention are located within one or more O-linkedglycosylation sites that do not naturally exist in wild type hGH. Inanother embodiment, mutations within the biologically active hGH mutantsof the present invention reside within as well as outside of one or moreO-linked glycosylation sites of the hGH mutants.

Representative wild type and mutant hGH polypeptides have sequences thatare selected from:

SEQ ID NO:19 (192 amino acid wild-type pituitary derived hGH comprisingan N-terminal methionine)mfptiplsrlfdnamlrahrlhqlafdtyqefeeayipkeqkysflqnpqtslcfsesiptpsnreetqqksnlellrisllliqswlepvqflrsvfanslvygasdsnvydllkdleegiqtlmgrledgsprtgqifkqtyskfdtnshnddallknygllycfrkdmdkvetflrivqcrsvegscgf SEQ ID NO:20 (191 amino acidwild-type pituitary derived hGH lacking an N-Terminal methionine)fptiplsrlfdnamlrahrlhqlafdtyqefeeayipkeqkysflqnpqtslcfsesiptpsnreetqqksnlellrisllliqswlepvqflrsvfanslvygasdsnvydllkdleegiqtlmgrledgsprtgqifkqtyskfdtnshnddallknygllycfrkdmdkvetflrivqcrsvegscgf SEQ ID NO:21 (wild type)MFPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKOTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF

The following are representative mutant peptide sequences correspondingto the region underlined in the wild type SEQ ID NO:21:LEDGSPTTGQIFKQTYS, LEDGSPTTAQIFKQTYS, LEDGSPTATQIFKQTYS,LEDGSPTQGAMFKQTYS, LEDGSPTQGAIFKQTYS, LEDGSPTQGQIFKQTYS,LEDGSPTTLYVFKQTYS, LEDGSPTINTIFKQTYS, LEDGSPTTVSIFKQTYS,LEDGSPRTGQIPTQTYS, LEDGSPRTGQIPTQAYS, LEDGSPTTLQIFKQTYS,LETETPRTGQIFKQTYS, LVTETPRTGQIFKQTYS, LETQSPRTGQIFKQTYS,LVTQSPRTGQIFKQTYS, LVTETPATGQIFKQTYS, LEDGSPTQGAMPKQTYS, andLEDGSPTTTQIFKQTYS.

In another exemplary embodiment, the peptide is a biologically activeIFN alpha mutant that includes one or more mutations at a sitecorresponding to T¹⁰⁶ of INF alpha 2, e.g. adjacent to or encompassingan amino acid position in IFN alpha wild type, which corresponds to oraligns with T¹⁰⁶ of INF alpha 2. Biologically active IFN alpha mutantsof the present invention include any IFN alpha polypeptide, in part orin whole, with one or more mutations that do not result in substantialor entire loss of its biological activity as it is measured by anysuitable functional assays known to one skilled in the art. In oneembodiment, mutations within the biologically active IFN alpha mutantsof the present invention are located within one or more O-linkedglycosylation sites that do not naturally exist in wild type IFN alpha.In another embodiment, mutations within the biologically active IFNalpha mutants of the present invention reside within as well as outsideof one or more O-linked glycosylation sites of the IFN alpha mutants.

A wild type and mutant IFN alpha polypeptide is shown below:

SEQ ID NO:22 (from wild type IFN 2b) ⁹⁸CVIQGVGVTETPLMKEDSIL¹¹⁷

Other appropriate O-linked glycosylation sequences for G-CSF andpeptides other than G-CSF can be determined by preparing a polypeptideincorporating a putative O-linked glycosylation site and submitting thatpolypeptide to suitable O-linked glycosylation conditions, therebyconfirming its ability to serve as an acceptor for a GalNac transferase.Moreover, as will be apparent to one of skill in the art, peptides thatinclude one or more mutation are within the scope of the presentinvention. The mutations are designed to allow the adjustment ofdesirable properties of the peptides, e.g., activity and number andposition of O- and/or N-linked glycosylation sites on the peptide.

Acquisition of Peptide Coding Sequences General Recombinant Technology

This invention relies on routine techniques in the field of recombinantgenetics. Basic texts disclosing the general methods of use in thisinvention include Sambrook and Russell, Molecular Cloning, A LaboratoryManual (3rd ed. 2001); Kriegler, Gene Transfer and Expression: ALaboratory Manual (1990); and Ausubel et al., eds., Current Protocols inMolecular Biology (1994).

For nucleic acids, sizes are given in either kilobases (kb) or basepairs (bp). These are estimates derived from agarose or acrylamide gelelectrophoresis, from sequenced nucleic acids, or from published DNAsequences. For proteins, sizes are given in kilodaltons (kDa) or aminoacid residue numbers. Proteins sizes are estimated from gelelectrophoresis, from sequenced proteins, from derived amino acidsequences, or from published protein sequences.

Oligonucleotides that are not commercially available can be chemicallysynthesized, e.g., according to the solid phase phosphoramidite triestermethod first described by Beaucage & Caruthers, Tetrahedron Lett. 22:1859-1862 (1981), using an automated synthesizer, as described in VanDevanter et. al., Nucleic Acids Res. 12: 6159-6168 (1984). Entire genescan also be chemically synthesized. Purification of oligonucleotides isperformed using any art-recognized strategy, e.g., native acrylamide gelelectrophoresis or anion-exchange HPLC as described in Pearson &Reanier, J. Chrom. 255: 137-149 (1983).

The sequence of the cloned wild-type peptide genes, polynucleotideencoding mutant peptides, and synthetic oligonucleotides can be verifiedafter cloning using, e.g., the chain termination method for sequencingdouble-stranded templates of Wallace et al., Gene 16: 21-26 (1981).

Cloning and Subcloning of a Wild-Type Peptide Coding Sequence

Numerous polynucleotide sequences encoding wild-type peptides have beendetermined and are available from a commercial supplier, e.g., humangrowth hormone, e.g., GenBank Accession Nos. NM 000515, NM 002059, NM022556, NM 022557, NM 022558, NM 022559, NM 022560, NM 022561, and NM022562.

The rapid progress in the studies of human genome has made possible acloning approach where a human DNA sequence database can be searched forany gene segment that has a certain percentage of sequence homology to aknown nucleotide sequence, such as one encoding a previously identifiedpeptide. Any DNA sequence so identified can be subsequently obtained bychemical synthesis and/or a polymerase chain reaction (PCR) techniquesuch as overlap extension method. For a short sequence, completely denovo synthesis may be sufficient; whereas further isolation of fulllength coding sequence from a human cDNA or genomic library using asynthetic probe may be necessary to obtain a larger gene.

Alternatively, a nucleic acid sequence encoding a peptide can beisolated from a human cDNA or genomic DNA library using standard cloningtechniques such as polymerase chain reaction (PCR), where homology-basedprimers can often be derived from a known nucleic acid sequence encodinga peptide. Most commonly used techniques for this purpose are describedin standard texts, e.g., Sambrook and Russell, supra.

cDNA libraries suitable for obtaining a coding sequence for a wild-typepeptide may be commercially available or can be constructed. The generalmethods of isolating mRNA, making cDNA by reverse transcription,ligating cDNA into a recombinant vector, transfecting into a recombinanthost for propagation, screening, and cloning are well known (see, e.g.,Gubler and Hoffman, Gene, 25: 263-269 (1983); Ausubel et al, supra).Upon obtaining an amplified segment of nucleotide sequence by PCR, thesegment can be further used as a probe to isolate the full-lengthpolynucleotide sequence encoding the wild-type peptide from the cDNAlibrary. A general description of appropriate procedures can be found inSambrook and Russell, supra.

A similar procedure can be followed to obtain a full length sequenceencoding a wild-type peptide, e.g., any one of the GenBank Accession Nosmentioned above, from a human genomic library. Human genomic librariesare commercially available or can be constructed according to variousart-recognized methods. In general, to construct a genomic library, theDNA is first extracted from an tissue where a peptide is likely found.The DNA is then either mechanically sheared or enzymatically digested toyield fragments of about 12-20 kb in length. The fragments aresubsequently separated by gradient centrifugation from polynucleotidefragments of undesired sizes and are inserted in bacteriophage λvectors. These vectors and phages are packaged in vitro. Recombinantphages are analyzed by plaque hybridization as described in Benton andDavis, Science, 196: 180-182 (1977). Colony hybridization is carried outas described by Grunstein et al., Proc. Natl. Acad. Sci. USA, 72:3961-3965 (1975).

Based on sequence homology, degenerate oligonucleotides can be designedas primer sets and PCR can be performed under suitable conditions (see,e.g., White et al., PCR Protocols: Current Methods and Applications,1993; Griffin and Griffin, PCR Technology, CRC Press Inc. 1994) toamplify a segment of nucleotide sequence from a cDNA or genomic library.Using the amplified segment as a probe, the full-length nucleic acidencoding a wild-type peptide is obtained.

Upon acquiring a nucleic acid sequence encoding a wild-type peptide, thecoding sequence can be subcloned into a vector, for instance, anexpression vector, so that a recombinant wild-type peptide can beproduced from the resulting construct. Further modifications to thewild-type peptide coding sequence, e.g., nucleotide substitutions, maybe subsequently made to alter the characteristics of the molecule.

Introducing Mutations into a Peptide Sequence

From an encoding polynucleotide sequence, the amino acid sequence of awild-type peptide can be determined. Subsequently, this amino acidsequence may be modified to alter the protein's glycosylation pattern,by introducing additional glycosylation site(s) at various locations inthe amino acid sequence.

Several types of protein glycosylation sites are well known in the art.For instance, in eukaryotes, N-linked glycosylation occurs on theasparagine of the consensus sequence Asn-X_(aa)-Ser/Thr, in which X_(aa)is any amino acid except proline (Kornfeld et al., Ann Rev Biochem54:631-664 (1985); Kukuruzinska et al., Proc. Natl. Acad. Sci. USA84:2145-2149 (1987); Herscovics et al., FASEB J 7:540-550 (1993); andOrlean, Saccharomyces Vol. 3 (1996)). O-linked glycosylation takes placeat serine or threonine residues (Tanner et al., Biochim. Biophys. Acta.906:81-91 (1987); and Hounsell et al., Glycoconj. J. 13:19-26 (1996)).Other glycosylation patterns are formed by linkingglycosylphosphatidylinositol to the carboxyl-terminal carboxyl group ofthe protein (Takeda et al., Trends Biochem. Sci. 20:367-371 (1995); andUdenfriend et al, Ann. Rev. Biochem. 64:593-591 (1995). Based on thisknowledge, suitable mutations can thus be introduced into a wild-typepeptide sequence to form new glycosylation sites.

Although direct modification of an amino acid residue within a peptidepolypeptide sequence may be suitable to introduce a new N-linked orO-linked glycosylation site, more frequently, introduction of a newglycosylation site is accomplished by mutating the polynucleotidesequence encoding a peptide. This can be achieved by using any of knownmutagenesis methods, some of which are discussed below. Exemplarymodifications to a G-CSF peptide include those illustrated in SEQ IDNO:5-18.

A variety of mutation-generating protocols are established and describedin the art. See, e.g., Zhang et al., Proc. Natl. Acad. Sci. USA, 94:4504-4509 (1997); and Stemmer, Nature, 370: 389-391 (1994). Theprocedures can be used separately or in combination to produce variantsof a set of nucleic acids, and hence variants of encoded polypeptides.Kits for mutagenesis, library construction, and otherdiversity-generating methods are commercially available.

Mutational methods of generating diversity include, for example,site-directed mutagenesis (Botstein and Shortle, Science, 229: 1193-1201(1985)), mutagenesis using uracil-containing templates (Kunkel, Proc.Natl. Acad. Sci. USA, 82: 488-492 (1985)), oligonucleotide-directedmutagenesis (Zoller and Smith, Nucl. Acids Res., 10: 6487-6500 (1982)),phosphorothioate-modified DNA mutagenesis (Taylor et al., Nucl. AcidsRes., 13: 8749-8764 and 8765-8787 (1985)), and mutagenesis using gappedduplex DNA (Kramer et al., Nucl. Acids Res., 12: 9441-9456 (1984)).

Other methods for generating mutations include point mismatch repair(Kramer et al., Cell, 38: 879-887 (1984)), mutagenesis usingrepair-deficient host strains (Carter et al., Nucl. Acids Res., 13:4431-4443 (1985)), deletion mutagenesis (Eghtedarzadeh and Henikoff,Nucl. Acids Res., 14: 5115 (1986)), restriction-selection andrestriction-purification (Wells et al., Phil. Trans. R. Soc. Lond. A,317: 415-423 (1986)), mutagenesis by total gene synthesis (Nambiar etal., Science, 223: 1299-1301 (1984)), double-strand break repair(Mandecki, Proc. Natl. Acad. Sci. USA, 83: 7177-7181 (1986)),mutagenesis by polynucleotide chain termination methods (U.S. Pat. No.5,965,408), and error-prone PCR (Leung et al., Biotechniques, 1: 11-15(1989)).

Modification of Nucleic Acids for Preferred Codon Usage in a HostOrganism

The polynucleotide sequence encoding a mutant peptide can be furtheraltered to coincide with the preferred codon usage of a particular host.For example, the preferred codon usage of one strain of bacterial cellscan be used to derive a polynucleotide that encodes a mutant peptide ofthe invention and includes the codons favored by this strain. Thefrequency of preferred codon usage exhibited by a host cell can becalculated by averaging frequency of preferred codon usage in a largenumber of genes expressed by the host cell (e.g., calculation service isavailable from web site of the Kazusa DNA Research Institute, Japan).This analysis is preferably limited to genes that are highly expressedby the host cell. U.S. Pat. No. 5,824,864, for example, provides thefrequency of codon usage by highly expressed genes exhibited bydicotyledonous plants and monocotyledonous plants.

At the completion of modification, the mutant peptide coding sequencesare verified by sequencing and are then subcloned into an appropriateexpression vector for recombinant production in the same manner as thewild-type peptides.

Expression and Purification of the Mutant Peptide

Following sequence verification, the mutant peptide of the presentinvention can be produced using routine techniques in the field ofrecombinant genetics, relying on the polynucleotide sequences encodingthe polypeptide disclosed herein.

Expression Systems

To obtain high-level expression of a nucleic acid encoding a mutantpeptide of the present invention, one typically subclones apolynucleotide encoding the mutant peptide into an expression vectorthat contains a strong promoter to direct transcription, atranscription/translation terminator and a ribosome binding site fortranslational initiation. Suitable bacterial promoters are well known inthe art and described, e.g., in Sambrook and Russell, supra, and Ausubelet al., supra. Bacterial expression systems for expressing the wild-typeor mutant peptide are available in, e.g., E. coli, Bacillus sp.,Salmonella, and Caulobacter. Kits for such expression systems arecommercially available. Eukaryotic expression systems for mammaliancells, yeast, and insect cells are well known in the art and are alsocommercially available. In one embodiment, the eukaryotic expressionvector is an adenoviral vector, an adeno-associated vector, or aretroviral vector.

The promoter used to direct expression of a heterologous nucleic aciddepends on the particular application. The promoter is optionallypositioned about the same distance from the heterologous transcriptionstart site as it is from the transcription start site in its naturalsetting. As is known in the art, however, some variation in thisdistance can be accommodated without loss of promoter function.

In addition to the promoter, the expression vector typically includes atranscription unit or expression cassette that contains all theadditional elements required for the expression of the mutant peptide inhost cells. A typical expression cassette thus contains a promoteroperably linked to the nucleic acid sequence encoding the mutant peptideand signals required for efficient polyadenylation of the transcript,ribosome binding sites, and translation termination. The nucleic acidsequence encoding the peptide is typically linked to a cleavable signalpeptide sequence to promote secretion of the peptide by the transformedcell. Such signal peptides include, among others, the signal peptidesfrom tissue plasminogen activator, insulin, and neuron growth factor,and juvenile hormone esterase of Heliothis virescens. Additionalelements of the cassette may include enhancers and, if genomic DNA isused as the structural gene, introns with functional splice donor andacceptor sites.

In addition to a promoter sequence, the expression cassette should alsocontain a transcription termination region downstream of the structuralgene to provide for efficient termination. The termination region may beobtained from the same gene as the promoter sequence or may be obtainedfrom different genes.

The particular expression vector used to transport the geneticinformation into the cell is not particularly critical. Any of theconventional vectors used for expression in eukaryotic or prokaryoticcells may be used. Standard bacterial expression vectors includeplasmids such as pBR322-based plasmids, pSKF, pET23D, and fusionexpression systems such as GST and LacZ. Epitope tags can also be addedto recombinant proteins to provide convenient methods of isolation,e.g., c-myc.

Expression vectors containing regulatory elements from eukaryoticviruses are typically used in eukaryotic expression vectors, e.g., SV40vectors, papilloma virus vectors, and vectors derived from Epstein-Barrvirus. Other exemplary eukaryotic vectors include pMSG, pAV009/A⁺,pMTO10/A⁺, pMAMneo-5, baculovirus pDSVE, and any other vector allowingexpression of proteins under the direction of the SV40 early promoter,SV40 later promoter, metallothionein promoter, murine mammary tumorvirus promoter, Rous sarcoma virus promoter, polyhedrin promoter, orother promoters shown effective for expression in eukaryotic cells.

In some exemplary embodiments the expression vector is chosen frompCWinl, pCWin2, pCWin2/MBP, pCWin2-MBP-SBD (pMS₃₉), andpCWin2-MBP-MCS-SBD (pMXS₃₉) as disclosed in co-owned U.S. Patentapplication filed Apr. 9, 2004 which is incorporated herein byreference.

Some expression systems have markers that provide gene amplificationsuch as thymidine kinase, hygromycin B phosphotransferase, anddihydrofolate reductase. Alternatively, high yield expression systemsnot involving gene amplification are also suitable, such as abaculovirus vector in insect cells, with a polynucleotide sequenceencoding the mutant peptide under the direction of the polyhedrinpromoter or other strong baculovirus promoters.

The elements that are typically included in expression vectors alsoinclude a replicon that functions in E. coli, a gene encoding antibioticresistance to permit selection of bacteria that harbor recombinantplasmids, and unique restriction sites in nonessential regions of theplasmid to allow insertion of eukaryotic sequences. The particularantibiotic resistance gene chosen is not critical, any of the manyresistance genes known in the art are suitable. The prokaryoticsequences are optionally chosen such that they do not interfere with thereplication of the DNA in eukaryotic cells, if necessary.

When periplasmic expression of a recombinant protein (e.g., a hgh mutantof the present invention) is desired, the expression vector furthercomprises a sequence encoding a secretion signal, such as the E. coliOppA (Periplasmic Oligopeptide Binding Protein) secretion signal or amodified version thereof, which is directly connected to 5′ of thecoding sequence of the protein to be expressed. This signal sequencedirects the recombinant protein produced in cytoplasm through the cellmembrane into the periplasmic space. The expression vector may furthercomprise a coding sequence for signal peptidase 1, which is capable ofenzymatically cleaving the signal sequence when the recombinant proteinis entering the periplasmic space. More detailed description forperiplasmic production of a recombinant protein can be found in, e.g.,Gray et al., Gene 39: 247-254 (1985), U.S. Pat. Nos. 6,160,089 and6,436,674.

As discussed above, a person skilled in the art will recognize thatvarious conservative substitutions can be made to any wild-type ormutant peptide or its coding sequence while still retaining thebiological activity of the peptide. Moreover, modifications of apolynucleotide coding sequence may also be made to accommodate preferredcodon usage in a particular expression host without altering theresulting amino acid sequence.

Transfection Methods

Standard transfection methods are used to produce bacterial, mammalian,yeast or insect cell lines that express large quantities of the mutantpeptide, which are then purified using standard techniques (see, e.g.,Colley et al., J. Biol. Chem. 264: 17619-17622 (1989); Guide to ProteinPurification, in Methods in Enzymology, vol. 182 (Deutscher, ed.,1990)). Transformation of eukaryotic and prokaryotic cells are performedaccording to standard techniques (see, e.g., Morrison, J. Bact. 132:349-351 (1977); Clark-Curtiss & Curtiss, Methods in Enzymology 101:347-362 (Wu et al., eds, 1983).

Any of the well-known procedures for introducing foreign nucleotidesequences into host cells may be used. These include the use of calciumphosphate transfection, polybrene, protoplast fusion, electroporation,liposomes, microinjection, plasma vectors, viral vectors and any of theother well known methods for introducing cloned genomic DNA, cDNA,synthetic DNA, or other foreign genetic material into a host cell (see,e.g., Sambrook and Russell, supra). It is only necessary that theparticular genetic engineering procedure used be capable of successfullyintroducing at least one gene into the host cell capable of expressingthe mutant peptide.

Detection of Expression of Mutant Peptide in Host Cells

After the expression vector is introduced into appropriate host cells,the transfected cells are cultured under conditions favoring expressionof the mutant peptide. The cells are then screened for the expression ofthe recombinant polypeptide, which is subsequently recovered from theculture using standard techniques (see, e.g., Scopes, ProteinPurification: Principles and Practice (1982); U.S. Pat. No. 4,673,641;Ausubel et al., supra; and Sambrook and Russell, supra).

Several general methods for screening gene expression are well knownamong those skilled in the art. First, gene expression can be detectedat the nucleic acid level. A variety of methods of specific DNA and RNAmeasurement using nucleic acid hybridization techniques are commonlyused (e.g., Sambrook and Russell, supra). Some methods involve anelectrophoretic separation (e.g., Southern blot for detecting DNA andNorthern blot for detecting RNA), but detection of DNA or RNA can becarried out without electrophoresis as well (such as by dot blot). Thepresence of nucleic acid encoding a mutant peptide in transfected cellscan also be detected by PCR or RT-PCR using sequence-specific primers.

Second, gene expression can be detected at the polypeptide level.Various immunological assays are routinely used by those skilled in theart to measure the level of a gene product, particularly usingpolyclonal or monoclonal antibodies that react specifically with amutant peptide of the present invention, such as a polypeptide havingthe amino acid sequence of SEQ ID NO:1-7, (e.g., Harlow and Lane,Antibodies, A Laboratory Manual, Chapter 14, Cold Spring Harbor, 1988;Kohler and Milstein, Nature, 256: 495-497 (1975)). Such techniquesrequire antibody preparation by selecting antibodies with highspecificity against the mutant peptide or an antigenic portion thereof.The methods of raising polyclonal and monoclonal antibodies are wellestablished and their descriptions can be found in the literature, see,e.g., Harlow and Lane, supra; Kohler and Milstein, Eur. J. Immunol., 6:511-519 (1976). More detailed descriptions of preparing antibody againstthe mutant peptide of the present invention and conducting immunologicalassays detecting the mutant peptide are provided in a later section.

Purification of Recombinantly Produced Mutant Peptide

Once the expression of a recombinant mutant peptide in transfected hostcells is confirmed, the host cells are then cultured in an appropriatescale for the purpose of purifying the recombinant polypeptide.

1. Purification of Recombinantly Produced Mutant Peptide from Bacteria

When the mutant peptides of the present invention are producedrecombinantly by transformed bacteria in large amounts, typically afterpromoter induction, although expression can be constitutive, theproteins may form insoluble aggregates. There are several protocols thatare suitable for purification of protein inclusion bodies. For example,purification of aggregate proteins (hereinafter referred to as inclusionbodies) typically involves the extraction, separation and/orpurification of inclusion bodies by disruption of bacterial cells, e.g.,by incubation in a buffer of about 100-150 μg/ml lysozyme and 0.1%Nonidet P40, a non-ionic detergent. The cell suspension can be groundusing a Polytron grinder (Brinkman Instruments, Westbury, N.Y.).Alternatively, the cells can be sonicated on ice. Alternate methods oflysing bacteria are described in Ausubel et al. and Sambrook andRussell, both supra, and will be apparent to those of skill in the art.

The cell suspension is generally centrifuged and the pellet containingthe inclusion bodies resuspended in buffer which does not dissolve butwashes the inclusion bodies, e.g., 20 mM Tris-HCl (pH 7.2), 1 mM EDTA,150 mM NaCl and 2% Triton-X 100, a non-ionic detergent. It may benecessary to repeat the wash step to remove as much cellular debris aspossible. The remaining pellet of inclusion bodies may be resuspended inan appropriate buffer (e.g., 20 mM sodium phosphate, pH 6.8, 150 mMNaCl). Other appropriate buffers will be apparent to those of skill inthe art.

Following the washing step, the inclusion bodies are solubilized by theaddition of a solvent that is both a strong hydrogen acceptor and astrong hydrogen donor (or a combination of solvents each having one ofthese properties). The proteins that formed the inclusion bodies maythen be renatured by dilution or dialysis with a compatible buffer.Suitable solvents include, but are not limited to, urea (from about 4 Mto about 8 M), formamide (at least about 80%, volume/volume basis), andguanidine hydrochloride (from about 4 M to about 8 M). Some solventsthat are capable of solubilizing aggregate-forming proteins, such as SDS(sodium dodecyl sulfate) and 70% formic acid, may be inappropriate foruse in this procedure due to the possibility of irreversibledenaturation of the proteins, accompanied by a lack of immunogenicityand/or activity. Although guanidine hydrochloride and similar agents aredenaturants, this denaturation is not irreversible and renaturation mayoccur upon removal (by dialysis, for example) or dilution of thedenaturant, allowing re-formation of the immunologically and/orbiologically active protein of interest. After solubilization, theprotein can be separated from other bacterial proteins by standardseparation techniques. For further description of purifying recombinantpeptide from bacterial inclusion body, see, e.g., Patra et al., ProteinExpression and Purification 18: 182-190 (2000).

Alternatively, it is possible to purify recombinant polypeptides, e.g. amutant peptide, from bacterial periplasm. Where the recombinant proteinis exported into the periplasm of the bacteria, the periplasmic fractionof the bacteria can be isolated by cold osmotic shock in addition toother methods known to those of skill in the art (see e.g., Ausubel etal., supra). To isolate recombinant proteins from the periplasm, thebacterial cells are centrifuged to form a pellet. The pellet isresuspended in a buffer containing 20% sucrose. To lyse the cells, thebacteria are centrifuged and the pellet is resuspended in ice-cold 5 mMMgSO₄ and kept in an ice bath for approximately 10 minutes. The cellsuspension is centrifuged and the supernatant decanted and saved. Therecombinant proteins present in the supernatant can be separated fromthe host proteins by standard separation techniques well known to thoseof skill in the art.

2. Standard Protein Separation Techniques for Purification

When a recombinant polypeptide, e.g., the mutant peptide of the presentinvention, is expressed in host cells in a soluble form, itspurification can follow the standard protein purification proceduredescribed below.

i. Solubility Fractionation

Often as an initial step, and if the protein mixture is complex, aninitial salt fractionation can separate many of the unwanted host cellproteins (or proteins derived from the cell culture media) from therecombinant protein of interest, e.g., a mutant peptide of the presentinvention. The preferred salt is ammonium sulfate. Ammonium sulfateprecipitates proteins by effectively reducing the amount of water in theprotein mixture. Proteins then precipitate on the basis of theirsolubility. The more hydrophobic a protein is, the more likely it is toprecipitate at lower ammonium sulfate concentrations. A typical protocolis to add saturated ammonium sulfate to a protein solution so that theresultant ammonium sulfate concentration is between 20-30%. This willprecipitate the most hydrophobic proteins. The precipitate is discarded(unless the protein of interest is hydrophobic) and ammonium sulfate isadded to the supernatant to a concentration known to precipitate theprotein of interest. The precipitate is then solubilized in buffer andthe excess salt removed if necessary, through either dialysis ordiafiltration. Other methods that rely on solubility of proteins, suchas cold ethanol precipitation, are well known to those of skill in theart and can be used to fractionate complex protein mixtures.

ii. Size Differential Filtration

Based on a calculated molecular weight, a protein of greater and lessersize can be isolated using ultrafiltration through membranes ofdifferent pore sizes (for example, Amicon or Millipore membranes). As afirst step, the protein mixture is ultrafiltered through a membrane witha pore size that has a lower molecular weight cut-off than the molecularweight of a protein of interest, e.g., a mutant peptide. The retentateof the ultrafiltration is then ultrafiltered against a membrane with amolecular cut off greater than the molecular weight of the protein ofinterest. The recombinant protein will pass through the membrane intothe filtrate. The filtrate can then be chromatographed as describedbelow.

iii. Column Chromatography

The proteins of interest (such as the mutant peptide of the presentinvention) can also be separated from other proteins on the basis oftheir size, net surface charge, hydrophobicity, or affinity for ligands.In addition, antibodies raised against peptide can be conjugated tocolumn matrices and the peptide immunopurified. All of these methods arewell known in the art.

It will be apparent to one of skill that chromatographic techniques canbe performed at any scale and using equipment from many differentmanufacturers (e.g., Pharmacia Biotech).

Immunoassays for Detection of Mutant Peptide Expression

To confirm the production of a recombinant mutant peptide, immunologicalassays may be useful to detect in a sample the expression of thepolypeptide. Immunological assays are also useful for quantifying theexpression level of the recombinant hormone. Antibodies against a mutantpeptide are necessary for carrying out these immunological assays.

Production of Antibodies against Mutant Peptide

Methods for producing polyclonal and monoclonal antibodies that reactspecifically with an immunogen of interest are known to those of skillin the art (see, e.g., Coligan, Current Protocols in ImmunologyWiley/Greene, N.Y., 1991; Harlow and Lane, Antibodies: A LaboratoryManual Cold Spring Harbor Press, NY, 1989; Stites et al. (eds.) Basicand Clinical Immunology (4th ed.) Lange Medical Publications, Los Altos,Calif., and references cited therein; Goding, Monoclonal Antibodies:Principles and Practice (2d ed.) Academic Press, New York, N.Y., 1986;and Kohler and Milstein Nature 256: 495-497, 1975). Such techniquesinclude antibody preparation by selection of antibodies from librariesof recombinant antibodies in phage or similar vectors (see, Huse et al.,Science 246: 1275-1281, 1989; and Ward et al., Nature 341: 544-546,1989).

In order to produce antisera containing antibodies with desiredspecificity, the polypeptide of interest (e.g., a mutant peptide of thepresent invention) or an antigenic fragment thereof can be used toimmunize suitable animals, e.g., mice, rabbits, or primates. A standardadjuvant, such as Freund's adjuvant, can be used in accordance with astandard immunization protocol. Alternatively, a synthetic antigenicpeptide derived from that particular polypeptide can be conjugated to acarrier protein and subsequently used as an immunogen.

The animal's immune response to the immunogen preparation is monitoredby taking test bleeds and determining the titer of reactivity to theantigen of interest. When appropriately high titers of antibody to theantigen are obtained, blood is collected from the animal and antiseraare prepared. Further fractionation of the antisera to enrich antibodiesspecifically reactive to the antigen and purification of the antibodiescan be performed subsequently, see, Harlow and Lane, supra, and thegeneral descriptions of protein purification provided above.

Monoclonal antibodies are obtained using various techniques familiar tothose of skill in the art. Typically, spleen cells from an animalimmunized with a desired antigen are immortalized, commonly by fusionwith a myeloma cell (see, Kohler and Milstein, Eur. J. Immunol.6:511-519, 1976). Alternative methods of immortalization include, e.g.,transformation with Epstein Barr Virus, oncogenes, or retroviruses, orother methods well known in the art. Colonies arising from singleimmortalized cells are screened for production of antibodies of thedesired specificity and affinity for the antigen, and the yield of themonoclonal antibodies produced by such cells may be enhanced by varioustechniques, including injection into the peritoneal cavity of avertebrate host.

Additionally, monoclonal antibodies may also be recombinantly producedupon identification of nucleic acid sequences encoding an antibody withdesired specificity or a binding fragment of such antibody by screeninga human B cell cDNA library according to the general protocol outlinedby Huse et al., supra. The general principles and methods of recombinantpolypeptide production discussed above are applicable for antibodyproduction by recombinant methods.

When desired, antibodies capable of specifically recognizing a mutantpeptide of the present invention can be tested for theircross-reactivity against the wild-type peptide and thus distinguishedfrom the antibodies against the wild-type protein. For instance,antisera obtained from an animal immunized with a mutant peptide can berun through a column on which a wild-type peptide is immobilized. Theportion of the antisera that passes through the column recognizes onlythe mutant peptide and not the wild-type peptide. Similarly, monoclonalantibodies against a mutant peptide can also be screened for theirexclusivity in recognizing only the mutant but not the wild-typepeptide.

Polyclonal or monoclonal antibodies that specifically recognize only themutant peptide of the present invention but not the wild-type peptideare useful for isolating the mutant protein from the wild-type protein,for example, by incubating a sample with a mutant peptide-specificpolyclonal or monoclonal antibody immobilized on a solid support.

Immunoassays for Detecting Mutant Peptide Expression

Once antibodies specific for a mutant peptide of the present inventionare available, the amount of the polypeptide in a sample, e.g., a celllysate, can be measured by a variety of immunoassay methods providingqualitative and quantitative results to a skilled artisan. For a reviewof immunological and immunoassay procedures in general see, e.g.,Stites, supra; U.S. Pat. Nos. 4,366,241; 4,376,110; 4,517,288; and4,837,168.

Labeling in Immunoassays

Immunoassays often utilize a labeling agent to specifically bind to andlabel the binding complex formed by the antibody and the target protein.The labeling agent may itself be one of the moieties comprising theantibody/target protein complex, or may be a third moiety, such asanother antibody, that specifically binds to the antibody/target proteincomplex. A label may be detectable by spectroscopic, photochemical,biochemical, immunochemical, electrical, optical or chemical means.Examples include, but are not limited to, magnetic beads (e.g.,Dynabeads™), fluorescent dyes (e.g., fluorescein isothiocyanate, Texasred, rhodamine, and the like), radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, or³²P), enzymes (e.g., horse radish peroxidase, alkaline phosphatase, andothers commonly used in an ELISA), and calorimetric labels such ascolloidal gold or colored glass or plastic (e.g., polystyrene,polypropylene, latex, etc.) beads.

In some cases, the labeling agent is a second antibody bearing adetectable label. Alternatively, the second antibody may lack a label,but it may, in turn, be bound by a labeled third antibody specific toantibodies of the species from which the second antibody is derived. Thesecond antibody can be modified with a detectable moiety, such asbiotin, to which a third labeled molecule can specifically bind, such asenzyme-labeled streptavidin.

Other proteins capable of specifically binding immunoglobulin constantregions, such as protein A or protein G, can also be used as the labelagents. These proteins are normal constituents of the cell walls ofstreptococcal bacteria. They exhibit a strong non-immunogenic reactivitywith immunoglobulin constant regions from a variety of species (see,generally, Kronval, et al. J. Immunol., 111: 1401-1406 (1973); andAkerstrom, et al., J. Immunol., 135: 2589-2542 (1985)).

Immunoassay Formats

Immunoassays for detecting a target protein of interest (e.g., a mutanthuman growth hormone) from samples may be either competitive ornoncompetitive. Noncompetitive immunoassays are assays in which theamount of captured target protein is directly measured. In one preferred“sandwich” assay, for example, the antibody specific for the targetprotein can be bound directly to a solid substrate where the antibody isimmobilized. It then captures the target protein in test samples. Theantibody/target protein complex thus immobilized is then bound by alabeling agent, such as a second or third antibody bearing a label, asdescribed above.

In competitive assays, the amount of target protein in a sample ismeasured indirectly by measuring the amount of an added (exogenous)target protein displaced (or competed away) from an antibody specificfor the target protein by the target protein present in the sample. In atypical example of such an assay, the antibody is immobilized and theexogenous target protein is labeled. Since the amount of the exogenoustarget protein bound to the antibody is inversely proportional to theconcentration of the target protein present in the sample, the targetprotein level in the sample can thus be determined based on the amountof exogenous target protein bound to the antibody and thus immobilized.

In some cases, western blot (immunoblot) analysis is used to detect andquantify the presence of a mutant peptide in the samples. The techniquegenerally comprises separating sample proteins by gel electrophoresis onthe basis of molecular weight, transferring the separated proteins to asuitable solid support (such as a nitrocellulose filter, a nylon filter,or a derivatized nylon filter) and incubating the samples with theantibodies that specifically bind the target protein. These antibodiesmay be directly labeled or alternatively may be subsequently detectedusing labeled antibodies (e.g., labeled sheep anti-mouse antibodies)that specifically bind to the antibodies against a mutant peptide.

Other assay formats include liposome immunoassays (LIA), which useliposomes designed to bind specific molecules (e.g., antibodies) andrelease encapsulated reagents or markers. The released chemicals arethen detected according to standard techniques (see, Monroe et al.,Amer. Clin. Prod. Rev., 5: 34-41 (1986)).

The Conjugates

In a representative aspect, the present invention provides aglycoconjugate between a peptide and a selected modifying group, inwhich the modifying group is conjugated to the peptide through aglycosyl linking group, e.g., an intact glycosyl linking group. Theglycosyl linking group is directly bound to an O-linked glycosylationsite on the peptide or, alternatively, it is bound to an O-linkedglycosylation site through one or more additional glycosyl residues.Methods of preparing the conjugates are set forth herein and in U.S.Pat. Nos. 5,876,980; 6,030,815; 5,728,554; 5,922,577; WO 98/31826;US2003180835; and WO 03/031464.

Exemplary peptides include an O-linked GalNAc residue that is bound tothe O-linked glycosylation site through the action of a GalNActransferase. The GalNAc itself may be the intact glycosyl linking group.The GalNAc may also be further elaborated by, for example, a Gal or Siaresidue, either of which can act as the intact glycosyl linking group.In representative embodiments, the O-linked saccharyl residue isGalNAc-X, GalNAc-Gal-Sia-X, or GalNAc-Gal-Gal-Sia-X, in which X is amodifying group.

In an exemplary embodiment, the peptide is a mutant peptide thatincludes an O-linked glycosylation site not present in the wild-typepeptide. The peptide is preferably O-glycosylated at the mutated sitewith a GalNAc residue. The discussion immediately preceding regardingthe structure of the saccharyl moiety is relevant here as well.

The link between the peptide and the selected moiety includes an intactglycosyl linking group interposed between the peptide and the modifyingmoiety. As discussed herein, the selected moiety is essentially anyspecies that can be attached to a saccharide unit, resulting in a“modified sugar” that is recognized by an appropriate transferaseenzyme, which appends the modified sugar onto the peptide. Thesaccharide component of the modified sugar, when interposed between thepeptide and a selected moiety, becomes an “intact glycosyl linkinggroup.” The glycosyl linking group is formed from any mono- oroligo-saccharide that, after modification with a selected moiety, is asubstrate for an appropriate transferase.

The conjugates of the invention will typically correspond to the generalstructure:

in which the symbols a, b, c, d and s represent a positive, non-zerointeger; and t is either 0 or a positive integer. The “agent” is atherapeutic agent, a bioactive agent, a detectable label, water-solublemoiety or the like. The “agent” can be a peptide, e.g, enzyme, antibody,antigen, etc. The linker can be any of a wide array of linking groups,infra. Alternatively, the linker may be a single bond or a “zero orderlinker.” The identity of the peptide is without limitation.

In an exemplary embodiment, the selected moiety is a water-solublepolymer, e.g., PEG, m-PEG, PPG, m-PPG, etc. The water-soluble polymer iscovalently attached to the peptide via a glycosyl linking group. Theglycosyl linking group is covalently attached to either an amino acidresidue or a glycosyl residue of the peptide. Alternatively, theglycosyl linking group is attached to one or more glycosyl units of aglycopeptide. The invention also provides conjugates in which theglycosyl linking group (e.g., GalNAc) is attached to an amino acidresidue (e.g., Thr or Ser).

In an exemplary embodiment, the protein is an interferon. Theinterferons are antiviral glycoproteins that, in humans, are secreted byhuman primary fibroblasts after induction with virus or double-strandedRNA. Interferons are of interest as therapeutics, e.g, antiviral agents(e.g., hepatitis B and C), antitumor agents (e.g., hepatocellularcarcinoma) and in the treatment of multiple sclerosis. For referencesrelevant to interferon-α, see, Asano, et al., Eur. J. Cancer, 27(Suppl4):S21-S25 (1991); Nagy, et al., Anticancer Research, 8(3):467-470(1988); Dron, et al., J. Biol. Regul. Homeost. Agents, 3(1):13-19(1989); Habib, et al., Am. Surg., 67(3):257-260 (3/2001); and Sugyiama,et al., Eur. J. Biochem., 217:921-927 (1993). For references discussingintefereon-β, see, e.g., Yu, et al., J. Neuroimmunol., 64(1):91-100(1996); Schmidt, J., J. Neurosci. Res., 65(1):59-67 (2001); Wender, etal., Folia Neuropathol., 39(2):91-93 (2001); Martin, et al., SpringerSemin. Immunopathol., 18(1):1-24 (1996); Takane, et al., J. Pharmacol.Exp. Ther., 294(2):746-752 (2000); Sburlati, et al., Biotechnol. Prog.,14:189-192 (1998); Dodd, et al., Biochimica et Biophysica Acta,787:183-187 (1984); Edelbaum, et al., J. Interferon Res., 12:449-453(1992); Conradt, et al., J. Biol. Chem., 262(30):14600-14605 (1987);Civas, et al., Eur. J. Biochem., 173:311-316 (1988); Demolder, et al.,J. Biotechnol., 32:179-189 (1994); Sedmak, et al., J. Interferon Res.,9(Suppl 1):S61-S65 (1989); Kagawa, et al., J. Biol. Chem.,263(33):17508-17515 (1988); Hershenson, et al., U.S. Pat. No. 4,894,330;Jayaram, et al., J. Interferon Res., 3(2):177-180 (1983); Menge, et al.,Develop. Biol. Standard., 66:391-401 (1987); Vonk, et al., J. InterferonRes., 3(2):169-175 (1983); and Adolf, et al., J. Interferon Res.,10:255-267 (1990).

In an exemplary interferon conjugate, interferon alpha, e.g., interferonalpha 2b and 2a, is conjugated to a water soluble polymer through anintact glycosyl linker.

In a further exemplary embodiment, the invention provides a conjugate ofhuman granulocyte colony stimulating factor (G-CSF). G-CSF is aglycoprotein that stimulates proliferation, differentiation andactivation of neutropoietic progenitor cells into functionally matureneutrophils. Injected G-CSF is rapidly cleared from the body. See, forexample, Nohynek, et al., Cancer Chemother. Pharmacol., 39:259-266(1997); Lord, et al., Clinical Cancer Research, 7(7):2085-2090(07/2001); Rotondaro, et al., Molecular Biotechnology, 11(2):117-128(1999); and Bönig, et al., Bone Marrow Transplantation, 28: 259-264(2001).

The present invention encompasses a method for the modification ofGM-CSF. GM-CSF is well known in the art as a cytokine produced byactivated T-cells, macrophages, endothelial cells, and stromalfibroblasts. GM-CSF primarily acts on the bone marrow to increase theproduction of inflammatory leukocytes, and further functions as anendocrine hormone to initiate the replenishment of neutrophils consumedduring inflammatory functions. Further GM-CSF is a macrophage-activatingfactor and promotes the differentiation of Lagerhans cells intodendritic cells. Like G-CSF, GM-CSF also has clinical applications inbone marrow replacement following chemotherapy

In addition to providing conjugates that are formed through anenzymatically added intact glycosyl linking group, the present inventionprovides conjugates that are highly homogenous in their substitutionpatterns. Using the methods of the invention, it is possible to formpeptide conjugates in which essentially all of the modified sugarmoieties across a population of conjugates of the invention are attachedto a structurally identical amino acid or glycosyl residue. Thus, in asecond aspect, the invention provides a peptide conjugate having apopulation of water-soluble polymer moieties, which are covalently boundto the peptide through an intact glycosyl linking group. In anotherconjugate of the invention, essentially each member of the population isbound via the glycosyl linking group to a glycosyl residue of thepeptide, and each glycosyl residue of the peptide to which the glycosyllinking group is attached has the same structure.

Also provided is a peptide conjugate having a population ofwater-soluble polymer moieties covalently bound thereto through aglycosyl linking group. In another embodiment, essentially every memberof the population of water soluble polymer moieties is bound to an aminoacid residue of the peptide via an intact glycosyl linking group, andeach amino acid residue having an intact glycosyl linking group attachedthereto has the same structure.

The present invention also provides conjugates analogous to thosedescribed above in which the peptide is conjugated to a therapeuticmoiety, diagnostic moiety, targeting moiety, toxin moiety or the likevia a glycosyl linking group. Each of the above-recited moieties can bea small molecule, natural polymer (e.g., polypeptide) or syntheticpolymer.

In a still further embodiment, the invention provides conjugates thatlocalize selectively in a particular tissue due to the presence of atargeting agent as a component of the conjugate. In an exemplaryembodiment, the targeting agent is a protein. Exemplary proteins includetransferrin (brain, blood pool), HS-glycoprotein (bone, brain, bloodpool), antibodies (brain, tissue with antibody-specific antigen, bloodpool), coagulation factors V-XII (damaged tissue, clots, cancer, bloodpool), serum proteins, e.g., α-acid glycoprotein, fetuin, α-fetalprotein (brain, blood pool), β2-glycoprotein (liver, atherosclerosisplaques, brain, blood pool), G-CSF, GM-CSF, M-CSF, and EPO (immunestimulation, cancers, blood pool, red blood cell overproduction,neuroprotection), albumin (increase in half-life), IL-2 and IFN-α.

In an exemplary targeted conjugate, interferon alpha 2β (IFN-α2β) isconjugated to transferrin via a bifunctional linker that includes anintact glycosyl linking group at each terminus of the PEG moiety (Scheme1). For example, one terminus of the PEG linker is functionalized withan intact sialic acid linker that is attached to transferrin and theother is functionalized with an intact O-linked GalNAc linker that isattached to IFN-α2β.

The conjugates of the invention can include glycosyl linking groups thatare mono- or multi-valent (e.g., antennary structures). Thus, conjugatesof the invention include both species in which a selected moiety isattached to a peptide via a monovalent glycosyl linking group. Alsoincluded within the invention are conjugates in which more than oneselected moiety is attached to a peptide via a multivalent linkinggroup.

The Methods

In addition to the conjugates discussed above, the present inventionprovides methods for preparing these and other conjugates. Moreover, theinvention provides methods of preventing, curing or ameliorating adisease state by administering a conjugate of the invention to a subjectat risk of developing the disease or a subject that has the disease.Additionally, the invention provides methods for targeting conjugates ofthe invention to a particular tissue or region of the body.

Thus, the invention provides a method of forming a covalent conjugatebetween a selected moiety and a peptide. In exemplary embodiments, theconjugate is formed between a water-soluble polymer, a therapeuticmoiety, targeting moiety or a biomolecule, and a glycosylated ornon-glycosylated peptide. The polymer, therapeutic moiety or biomoleculeis conjugated to the peptide via a glycosyl linking group, which isinterposed between, and covalently linked to both the peptide and themodifying group (e.g. water-soluble polymer). The method includescontacting the peptide with a mixture containing a modified sugar and aglycosyltransferase for which the modified sugar is a substrate. Thereaction is conducted under conditions appropriate to form a covalentbond between the modified sugar and the peptide. The sugar moiety of themodified sugar is preferably selected from nucleotide sugars, activatedsugars and sugars, which are neither nucleotides nor activated.

The acceptor peptide (O-glycosylated or non-glycosylated) is typicallysynthesized de novo, or recombinantly expressed in a prokaryotic cell(e.g., bacterial cell, such as E. coli) or in a eukaryotic cell such asa mammalian, yeast, insect, fungal or plant cell. The peptide can beeither a full-length protein or a fragment. Moreover, the peptide can bea wild type or mutated peptide. In an exemplary embodiment, the peptideincludes a mutation that adds one or more N- or O-linked glycosylationsites to the peptide sequence.

In an exemplary embodiment, the peptide is O-glycosylated andfunctionalized with a water-soluble polymer in the following manner. Thepeptide is either produced with an available amino acid glycosylationsite or, if glycosylated, the glycosyl moiety is trimmed off to exposedthe amino acid. For example, GalNAc is added to a serine or threonineand the galactosylated peptide is sialylated with a sialicacid-modifying group cassette using ST6Gal-1. Alternatively, thegalactosylated peptide is galactosylated using Core-1-GalT-1 and theproduct is sialylated with a sialic acid-modifying group cassette usingST3GalT1. An exemplary conjugate according to this method has thefollowing linkages: Thr-α-1-GalNAc-β-1,3-Gal-α-2,3-Sia*, in which Sia*is the sialic acid-modifying group cassette.

In the methods of the invention, such as that set forth above, usingmultiple enzymes and saccharyl donors, the individual glycosylationsteps may be performed separately, or combined in a “single pot”reaction. For example, in the three enzyme reaction set forth above theGalNAc tranferase, GalT and SiaT and their donors may be combined in asingle vessel. Alternatively, the GalNAc reaction can be performed aloneand both the GalT and SiaT and the appropriate saccharyl donors added asa single step. Another mode of running the reactions involves addingeach enzyme and an appropriate donor sequentially and conducting thereaction in a “single pot” motif. Combinations of each of the methodsset forth above are of use in preparing the compounds of the invention.

In the conjugates of the invention, the Sia-modifying group cassette canbe linked to the Gal in an α-2,6, or α-2,3 linkage.

For example, in one embodiment, G-CSF is expressed in a mammalian systemand modified by treatment of sialidase to trim back terminal sialic acidresidues, followed by PEGylation using ST3Gal3 and a donor of PEG-sialicacid.

The method of the invention also provides for modification ofincompletely glycosylated peptides that are produced recombinantly. Manyrecombinantly produced glycoproteins are incompletely glycosylated,exposing carbohydrate residues that may have undesirable properties,e.g., immunogenicity, recognition by the RES. Employing a modified sugarin a method of the invention, the peptide can be simultaneously furtherglycosylated and derivatized with, e.g., a water-soluble polymer,therapeutic agent, or the like. The sugar moiety of the modified sugarcan be the residue that would properly be conjugated to the acceptor ina fully glycosylated peptide, or another sugar moiety with desirableproperties.

Peptides modified by the methods of the invention can be synthetic orwild-type peptides or they can be mutated peptides, produced by methodsknown in the art, such as site-directed mutagenesis. Glycosylation ofpeptides is typically either N-linked or O-linked. An exemplaryN-linkage is the attachment of the modified sugar to the side chain ofan asparagine residue. The tripeptide sequences asparagine-X-serine andasparagine-X-threonine, where X is any amino acid except proline, arethe recognition sequences for enzymatic attachment of a carbohydratemoiety to the asparagine side chain. Thus, the presence of either ofthese tripeptide sequences in a polypeptide creates a potentialglycosylation site. O-linked glycosylation refers to the attachment ofone sugar (e.g., N-acetylgalactosamine, galactose, mannose, GlcNAc,glucose, fucose or xylose) to the hydroxy side chain of a hydroxyaminoacid, preferably serine or threonine, although unusual or non-naturalamino acids, e.g., 5-hydroxyproline or 5-hydroxylysine may also be used.

Moreover, in addition to peptides, the methods of the present inventioncan be practiced with other biological structures (e.g., glycolipids,lipids, sphingoids, ceramides, whole cells, and the like, containing anO-linked glycosylation site).

Addition of glycosylation sites to a peptide or other structure isconveniently accomplished by altering the amino acid sequence such thatit contains one or more glycosylation sites. The addition may also bemade by the incorporation of one or more species presenting an —OHgroup, preferably serine or threonine residues, within the sequence ofthe peptide (for O-linked glycosylation sites). The addition may be madeby mutation or by full chemical synthesis of the peptide. The peptideamino acid sequence is preferably altered through changes at the DNAlevel, particularly by mutating the DNA encoding the peptide atpreselected bases such that codons are generated that will translateinto the desired amino acids. The DNA mutation(s) are preferably madeusing methods known in the art.

In an exemplary embodiment, the glycosylation site is added by shufflingpolynucleotides. Polynucleotides encoding a candidate peptide can bemodulated with DNA shuffling protocols. DNA shuffling is a process ofrecursive recombination and mutation, performed by random fragmentationof a pool of related genes, followed by reassembly of the fragments by apolymerase chain reaction-like process. See, e.g., Stemmer, Proc. Natl.Acad. Sci. USA 91:10747-10751 (1994); Stemmer, Nature 370:389-391(1994); and U.S. Pat. Nos. 5,605,793, 5,837,458, 5,830,721 and5,811,238.

The present invention also provides means of adding (or removing) one ormore selected glycosyl residues to a peptide, after which a modifiedsugar is conjugated to at least one of the selected glycosyl residues ofthe peptide. The present embodiment is useful, for example, when it isdesired to conjugate the modified sugar to a selected glycosyl residuethat is either not present on a peptide or is not present in a desiredamount. Thus, prior to coupling a modified sugar to a peptide, theselected glycosyl residue is conjugated to the peptide by enzymatic orchemical coupling. In another embodiment, the glycosylation pattern of aglycopeptide is altered prior to the conjugation of the modified sugarby the removal of a carbohydrate residue from the glycopeptide. See, forexample WO 98/31826.

Addition or removal of any carbohydrate moieties present on theglycopeptide is accomplished either chemically or enzymatically.Chemical deglycosylation is preferably brought about by exposure of thepolypeptide variant to the compound trifluoromethanesulfonic acid, or anequivalent compound. This treatment results in the cleavage of most orall sugars except the linking sugar (N-acetylglucosamine orN-acetylgalactosamine), while leaving the peptide intact. Chemicaldeglycosylation is described by Hakimuddin et al., Arch. Biochem.Biophys. 259: 52 (1987) and by Edge et al., Anal Biochem. 118: 131(1981). Enzymatic cleavage of carbohydrate moieties on polypeptidevariants can be achieved by the use of a variety of endo- andexo-glycosidases as described by Thotakura et al., Meth. Enzymol. 138:350 (1987).

Chemical addition of glycosyl moieties is carried out by anyart-recognized method. Enzymatic addition of sugar moieties ispreferably achieved using a modification of the methods set forthherein, substituting native glycosyl units for the modified sugars usedin the invention. Other methods of adding sugar moieties are disclosedin U.S. Pat. Nos. 5,876,980, 6,030,815, 5,728,554, and 5,922,577.

Exemplary attachment points for selected glycosyl residue include, butare not limited to: (a) consensus sites for N-linked glycosylation, andsites for O-linked glycosylation; (b) terminal glycosyl moieties thatare acceptors for a glycosyltransferase; (c) arginine, asparagine andhistidine; (d) free carboxyl groups; (e) free sulfhydryl groups such asthose of cysteine; (f) free hydroxyl groups such as those of serine,threonine, or hydroxyproline; (g) aromatic residues such as those ofphenylalanine, tyrosine, or tryptophan; or (h) the amide group ofglutamine. Exemplary methods of use in the present invention aredescribed in WO 87/05330 published Sep. 11, 1987, and in Aplin andWriston, CRC CRIT. REV. BIOCHEM., pp. 259-306 (1981).

In one embodiment, the invention provides a method for linking two ormore peptides through a linking group. The linking group is of anyuseful structure and may be selected from straight- and branched-chainstructures. Preferably, each terminus of the linker, which is attachedto a peptide, includes a modified sugar (i.e., a nascent intact glycosyllinking group).

In an exemplary method of the invention, two peptides are linkedtogether via a linker moiety that includes a PEG linker. The constructconforms to the general structure set forth in the cartoon above. Asdescribed herein, the construct of the invention includes two intactglycosyl linking groups (i.e., s+t=1). The focus on a PEG linker thatincludes two glycosyl groups is for purposes of clarity and should notbe interpreted as limiting the identity of linker arms of use in thisembodiment of the invention.

Thus, a PEG moiety is functionalized at a first terminus with a firstglycosyl unit and at a second terminus with a second glycosyl unit. Thefirst and second glycosyl units are preferably substrates for differenttransferases, allowing orthogonal attachment of the first and secondpeptides to the first and second glycosylunits, respectively. Inpractice, the (glycosyl)¹-PEG-(glycosyl)² linker is contacted with thefirst peptide and a first transferase for which the first glycosyl unitis a substrate, thereby forming (peptide)¹-(glycosyl)¹-PEG-(glycosyl)².Transferase and/or unreacted peptide is then optionally removed from thereaction mixture. The second peptide and a second transferase for whichthe second glycosyl unit is a substrate are added to the(peptide)¹-(glycosyl)¹-PEG-(glycosyl)² conjugate, forming(peptide)¹-(glycosyl)¹-PEG-(glycosyl)²-(peptide)²; at least one of theglycosyl residues is either directly or indirectly O-linked. Those ofskill in the art will appreciate that the method outlined above is alsoapplicable to forming conjugates between more than two peptides by, forexample, the use of a branched PEG, dendrimer, poly(amino acid),polsaccharide or the like

In an exemplary embodiment, interferon alpha 2β (IFN-α 2β) is conjugatedto transferrin via a bifunctional linker that includes an intactglycosyl linking group at each terminus of the PEG moiety (Scheme 1).The IFN conjugate has an in vivo half-life that is increased over thatof IFN alone by virtue of the greater molecular sized of the conjugate.Moreover, the conjugation of IFN to transferrin serves to selectivelytarget the conjugate to the brain. For example, one terminus of the PEGlinker is functionalized with a CMP sialic acid and the other isfunctionalized with an UDP GalNAc. The linker is combined with IFN inthe presence of a GalNAc transferase, resulting in the attachment of theGalNAc of the linker arm to a serine and/or threonine residue on theIFN.

The processes described above can be carried through as many cycles asdesired, and is not limited to forming a conjugate between two peptideswith a single linker. Moreover, those of skill in the art willappreciate that the reactions functionalizing the intact glycosyllinking groups at the termini of the PEG (or other) linker with thepeptide can occur simultaneously in the same reaction vessel, or theycan be carried out in a step-wise fashion. When the reactions arecarried out in a step-wise manner, the conjugate produced at each stepis optionally purified from one or more reaction components (e.g.,enzymes, peptides).

A still further exemplary embodiment is set forth in Scheme 2. Scheme 2shows a method of preparing a conjugate that targets a selected protein,e.g., GM-CSF, to bone and increases the circulatory half-life of theselected protein.

in which G is a glycosyl residue on an activated sugar moiety (e.g.,sugar nucleotide), which is converted to an intact glycosyl linker groupin the conjugate. When s is greater than 0, L is a saccharyl linkinggroup such as GalNAc, or GalNAc-Gal.

The use of reactive derivatives of PEG (or other linkers) to attach oneor more peptide moieties to the linker is within the scope of thepresent invention. The invention is not limited by the identity of thereactive PEG analogue. Many activated derivatives ofpoly(ethyleneglycol) are available commercially and in the literature.It is well within the abilities of one of skill to choose, andsynthesize if necessary, an appropriate activated PEG derivative withwhich to prepare a substrate useful in the present invention. See,Abuchowski et al. Cancer Biochem. Biophys., 7: 175-186 (1984);Abuchowski et al., J. Biol. Chem., 252: 3582-3586 (1977); Jackson etal., Anal. Biochem., 165: 114-127 (1987); Koide et al., Biochem Biophys.Res. Commun., 111: 659-667 (1983)), tresylate (Nilsson et al., MethodsEnzymol., 104: 56-69 (1984); Delgado et al., Biotechnol. Appl. Biochem.,12: 119-128 (1990)); N-hydroxysuccinimide derived active esters(Buckmann et al., Makromol. Chem., 182: 1379-1384 (1981); Joppich etal., Makromol. Chem., 180: 1381-1384 (1979); Abuchowski et al., CancerBiochem. Biophys., 7: 175-186 (1984); Katre et al., Proc. Natl. Acad.Sci. U.S.A., 84: 1487-1491 (1987); Kitamura et al., Cancer Res., 51:4310-4315 (1991); Boccu et al., Z. Naturforsch., 38C: 94-99 (1983),carbonates (Zalipsky et al., POLY(ETHYLENE GLYCOL) CHEMISTRY:BIOTECHNICAL AND BIOMEDICAL APPLICATIONS, Harris, Ed., Plenum Press, NewYork, 1992, pp. 347-370; Zalipsky et al., Biotechnol. Appl. Biochem.,15: 100-114 (1992); Veronese et al., Appl. Biochem. Biotech., 11:141-152 (1985)), imidazolyl formates (Beauchamp et al., Anal. Biochem.,131: 25-33 (1983); Berger et al., Blood, 71: 1641-1647 (1988)),4-dithiopyridines (Woghiren et al., Bioconjugate Chem., 4: 314-318(1993)), isocyanates (Byun et al., ASAIO Journal, M649-M-653 (1992)) andepoxides (U.S. Pat. No. 4,806,595, issued to Noishiki et al., (1989).Other linking groups include the urethane linkage between amino groupsand activated PEG. See, Veronese, et al., Appl Biochem. Biotechnol., 11:141-152 (1985).

In another exemplary embodiment in which a reactive PEG derivative isutilized, the invention provides a method for extending theblood-circulation half-life of a selected peptide, in essence targetingthe peptide to the blood pool, by conjugating the peptide to a syntheticor natural polymer of a size sufficient to retard the filtration of theprotein by the glomerulus (e.g., albumin). See, Scheme 3. Thisembodiment of the invention is illustrated in Scheme in which G-CSF isconjugated to albumin via a PEG linker using a combination of chemicaland enzymatic modification.

Thus, as shown in Scheme 3, a residue (e.g., amino acid side chain) ofalbumin is modified with a reactive PEG derivative, such asX-PEG-(CMP-sialic acid), in which X is an activating group (e.g, activeester, isothiocyanate, etc). The PEG derivative and G-CSF are combinedand contacted with a transferase for which CMP-sialic acid is asubstrate. In a further illustrative embodiment, an ε-amine of lysine isreacted with the N-hydroxysuccinimide ester of the PEG-linker to formthe albumin conjugate. The CMP-sialic acid of the linker isenzymatically conjugated to an appropriate residue on GCSF, e.g, Gal, orGalNAc thereby forming the conjugate. Those of skill will appreciatethat the above-described method is not limited to the reaction partnersset forth. Moreover, the method can be practiced to form conjugates thatinclude more than two protein moieties by, for example, utilizing abranched linker having more than two termini.

Modified Sugars

Modified glycosyl donor species (“modified sugars”) are preferablyselected from modified sugar nucleotides, activated modified sugars andmodified sugars that are simple saccharides that are neither nucleotidesnor activated. Any desired carbohydrate structure can be added to apeptide using the methods of the invention. Typically, the structurewill be a monosaccharide, but the present invention is not limited tothe use of modified monosaccharide sugars; oligosaccharides andpolysaccharides are useful as well.

The modifying group is attached to a sugar moiety by enzymatic means,chemical means or a combination thereof, thereby producing a modifiedsugar. The sugars are substituted at any position that allows for theattachment of the modifying moiety, yet which still allows the sugar tofunction as a substrate for the enzyme used to ligate the modified sugarto the peptide. In another embodiment, when sialic acid is the sugar,the sialic acid is substituted with the modifying group at either the9-position on the pyruvyl side chain or at the 5-position on the aminemoiety that is normally acetylated in sialic acid.

In certain embodiments of the present invention, a modified sugarnucleotide is utilized to add the modified sugar to the peptide.Exemplary sugar nucleotides that are used in the present invention intheir modified form include nucleotide mono-, di- or triphosphates oranalogs thereof. In another embodiment, the modified sugar nucleotide isselected from a UDP-glycoside, CMP-glycoside, or a GDP-glycoside. Evenmore preferably, the modified sugar nucleotide is selected from anUDP-galactose, UDP-galactosamine, UDP-glucose, UDP-glucosamine,GDP-mannose, GDP-fucose, CMP-sialic acid, or CMP-NeuAc. N-acetylaminederivatives of the sugar nucletides are also of use in the method of theinvention.

The invention also provides methods for synthesizing a modified peptideusing a modified sugar, e.g., modified-galactose, -fucose, -GalNAc and-sialic acid. When a modified sialic acid is used, either asialyltransferase or a trans-sialidase (for α-2,3-linked sialic acidonly) can be used in these methods.

In other embodiments, the modified sugar is an activated sugar.Activated modified sugars, which are useful in the present invention aretypically glycosides which have been synthetically altered to include anactivated leaving group. As used herein, the term “activated leavinggroup” refers to those moieties, which are easily displaced inenzyme-regulated nucleophilic substitution reactions. Many activatedsugars are known in the art. See, for example, Vocadlo et al., InCARBOHYDRATE CHEMISTRY AND BIOLOGY, Vol. 2, Ernst et al. Ed., Wiley-VCHVerlag: Weinheim, Germany, 2000; Kodama et al., Tetrahedron Lett. 34:6419 (1993); Lougheed, et al., J. Biol. Chem. 274: 37717 (1999)).

Examples of activating groups (leaving groups) include fluoro, chloro,bromo, tosylate ester, mesylate ester, triflate ester and the like.Preferred activated leaving groups, for use in the present invention,are those that do not significantly sterically encumber the enzymatictransfer of the glycoside to the acceptor. Accordingly, preferredembodiments of activated glycoside derivatives include glycosylfluorides and glycosyl mesylates, with glycosyl fluorides beingparticularly preferred. Among the glycosyl fluorides, α-galactosylfluoride, α-mannosyl fluoride, α-glucosyl fluoride, α-fucosyl fluoride,α-xylosyl fluoride, α-sialyl fluoride, α-N-acetylglucosaminyl fluoride,α-N-acetylgalactosaminyl fluoride, β-galactosyl fluoride, β-mannosylfluoride, β-glucosyl fluoride, β-fucosyl fluoride, β-xylosyl fluoride,β-sialyl fluoride, β-N-acetylglucosaminyl fluoride andβ-N-acetylgalactosaminyl fluoride are most preferred.

By way of illustration, glycosyl fluorides can be prepared from the freesugar by first acetylating the sugar and then treating it withHF/pyridine. This generates the thermodynamically most stable anomer ofthe protected (acetylated) glycosyl fluoride (i.e., the α-glycosylfluoride). If the less stable anomer (i.e., the β-glycosyl fluoride) isdesired, it can be prepared by converting the peracetylated sugar withHBr/HOAc or with HCl to generate the anomeric bromide or chloride. Thisintermediate is reacted with a fluoride salt such as silver fluoride togenerate the glycosyl fluoride. Acetylated glycosyl fluorides may bedeprotected by reaction with mild (catalytic) base in methanol (e.g.NaOMe/MeOH). In addition, many glycosyl fluorides are commerciallyavailable.

Other activated glycosyl derivatives can be prepared using conventionalmethods known to those of skill in the art. For example, glycosylmesylates can be prepared by treatment of the fully benzylatedhemiacetal form of the sugar with mesyl chloride, followed by catalytichydrogenation to remove the benzyl groups.

In a further exemplary embodiment, the modified sugar is anoligosaccharide having an antennary structure. In another embodiment,one or more of the termini of the antennae bear the modifying moiety.When more than one modifying moiety is attached to an oligosaccharidehaving an antennary structure, the oligosaccharide is useful to“amplify” the modifying moiety; each oligosaccharide unit conjugated tothe peptide attaches multiple copies of the modifying group to thepeptide. The general structure of a typical conjugate of the inventionas set forth in the drawing above, encompasses multivalent speciesresulting from preparing a conjugate of the invention utilizing anantennary structure. Many antennary saccharide structures are known inthe art, and the present method can be practiced with them withoutlimitation.

Exemplary modifying groups are discussed below. The modifying groups canbe selected for their ability to impart to a peptide one or moredesirable property. Exemplary properties include, but are not limitedto, enhanced pharmacokinetics, enhanced pharmacodynamics, improvedbiodistribution, providing a polyvalent species, improved watersolubility, enhanced or diminished lipophilicity, and tissue targeting.

Water-Soluble Polymers

Many water-soluble polymers are known to those of skill in the art andare useful in practicing the present invention. The term water-solublepolymer encompasses species such as saccharides (e.g., dextran, amylose,hyalouronic acid, poly(sialic acid), heparans, heparins, etc.);poly(amino acids), e.g., poly(aspartic acid) and poly(glutamic acid);nucleic acids; synthetic polymers (e.g., poly(acrylic acid),poly(ethers), e.g., poly(ethylene glycol); peptides, proteins, and thelike. The present invention may be practiced with any water-solublepolymer with the sole limitation that the polymer must include a pointat which the remainder of the conjugate can be attached.

Methods for activation of polymers can also be found in WO 94/17039,U.S. Pat. No. 5,324,844, WO 94/18247, WO 94/04193, U.S. Pat. No.5,219,564, U.S. Pat. No. 5,122,614, WO 90/13540, U.S. Pat. No.5,281,698, and more WO 93/15189, and for conjugation between activatedpolymers and peptides, e.g. Coagulation Factor VIII (WO 94/15625),hemoglobin (WO 94/09027), oxygen carrying molecule (U.S. Pat. No.4,412,989), ribonuclease and superoxide dismutase (Veronese at al., App.Biochem. Biotech. 11: 141-45 (1985)).

Preferred water-soluble polymers are those in which a substantialproportion of the polymer molecules in a sample of the polymer are ofapproximately the same molecular weight; such polymers are“homodisperse.”

The present invention is further illustrated by reference to apoly(ethylene glycol) conjugate. Several reviews and monographs on thefunctionalization and conjugation of PEG are available. See, forexample, Harris, Macronol. Chem. Phys. C25: 325-373 (1985); Scouten,Methods in Enzymology 135: 30-65 (1987); Wong et al., Enzyme Microb.Technol. 14: 866-874 (1992); Delgado et al., Critical Reviews inTherapeutic Drug Carrier Systems 9: 249-304 (1992); Zalipsky,Bioconjugate Chem. 6: 150-165 (1995); and Bhadra, et al., Pharmazie,57:5-29 (2002). Routes for preparing reactive PEG molecules and formingconjugates using the reactive molecules are known in the art. Forexample, U.S. Pat. No. 5,672,662 discloses a water soluble andisolatable conjugate of an active ester of a polymer acid selected fromlinear or branched poly(alkylene oxides), poly(oxyethylated polyols),poly(olefinic alcohols), and poly(acrylomorpholine).

U.S. Pat. No. 6,376,604 sets forth a method for preparing awater-soluble 1-benzotriazolylcarbonate ester of a water-soluble andnon-peptidic polymer by reacting a terminal hydroxyl of the polymer withdi(1-benzotriazoyl)carbonate in an organic solvent. The active ester isused to form conjugates with a biologically active agent such as aprotein or peptide.

WO 99/45964 describes a conjugate comprising a biologically active agentand an activated water soluble polymer comprising a polymer backbonehaving at least one terminus linked to the polymer backbone through astable linkage, wherein at least one terminus comprises a branchingmoiety having proximal reactive groups linked to the branching moiety,in which the biologically active agent is linked to at least one of theproximal reactive groups. Other branched poly(ethylene glycols) aredescribed in WO 96/21469, U.S. Pat. No. 5,932,462 describes a conjugateformed with a branched PEG molecule that includes a branched terminusthat includes reactive functional groups. The free reactive groups areavailable to react with a biologically active species, such as a proteinor peptide, forming conjugates between the poly(ethylene glycol) and thebiologically active species. U.S. Pat. No. 5,446,090 describes abifunctional PEG linker and its use in forming conjugates having apeptide at each of the PEG linker termini.

Conjugates that include degradable PEG linkages are described in WO99/34833; and WO 99/14259, as well as in U.S. Pat. No. 6,348,558. Suchdegradable linkages are applicable in the present invention.

The art-recognized methods of polymer activation set forth above are ofuse in the context of the present invention in the formation of thebranched polymers set forth herein and also for the conjugation of thesebranched polymers to other species, e.g., sugars, sugar nucleotides andthe like.

Exemplary poly(ethylene glycol) molecules of use in the inventioninclude, but are not limited to, those having the formula:

in which R⁸ is H, OH, NH₂, substituted or unsubstituted alkyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted heteroalkyl, e.g., acetal, OHC—, H₂N—(CH₂)_(q)—,HS—(CH₂)_(q), or —(CH₂)_(q)C(Y)Z¹. The index “e” represents an integerfrom 1 to 2500. The indices b, d, and q independently represent integersfrom 0 to 20. The symbols Z and Z¹ independently represent OH, NH₂,leaving groups, e.g., imidazole, p-nitrophenyl, HOBT, tetrazole, halide,S—R⁹, the alcohol portion of activated esters; —(CH₂)_(p)C(Y¹)V, or—(CH₂)_(p)U(CH₂)_(n)C(Y¹)_(v). The symbol Y represents H(2), ═O, ═S,═N—R¹⁰. The symbols X, Y, Y¹, A¹, and U independently represent themoieties O, S, N—R¹¹. The symbol V represents OH, NH₂, halogen, S—R¹²,the alcohol component of activated esters, the amine component ofactivated amides, sugar-nucleotides, and proteins. The indices p, q, sand v are members independently selected from the integers from 0 to 20.The symbols R⁹, R¹⁰, R¹¹ and R¹² independently represent H, substitutedor unsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted aryl, substituted or unsubstitutedheterocycloalkyl and substituted or unsubstituted heteroaryl.

In other exemplary embodiments, the poly(ethylene glycol) molecule isselected from the following:

The poly(ethylene glycol) useful in forming the conjugate of theinvention is either linear or branched. Branched poly(ethylene glycol)molecules suitable for use in the invention include, but are not limitedto, those described by the following formula:

in which R⁸ and R^(8′) are members independently selected from thegroups defined for R⁸, above. A¹ and A² are members independentlyselected from the groups defined for A¹, above. The indices e, f, o, andq are as described above. Z and Y are as described above. X¹ and X^(1′)are members independently selected from S, SC(O)NH, HNC(O)S, SC(O)O, O,NH, NHC(O), (O)CNH and NHC(O)O, OC(O)NH.

In other exemplary embodiments, the branched PEG is based upon acysteine, serine or di-lysine core. Thus, further exemplary branchedPEGs include:

In yet another embodiment, the branched PEG moiety is based upon atri-lysine peptide. The tri-lysine can be mono-, di-, tri-, ortetra-PEG-ylated. Exemplary species according to this embodiment havethe formulae:

in which e, f and f′ are independently selected integers from 1 to 2500;and q, q′ and q″ are independently selected integers from 1 to 20.

In exemplary embodiments of the invention, the PEG is m-PEG (5 kD, 10kD, or 20 kD). An exemplary branched PEG species is a serine- orcysteine-(m-PEG)₂ in which the m-PEG is a 20 kD m-PEG.

As will be apparent to those of skill, the branched polymers of use inthe invention include variations on the themes set forth above. Forexample the di-lysine-PEG conjugate shown above can include threepolymeric subunits, the third bonded to the α-amine shown as unmodifiedin the structure above. Similarly, the use of a tri-lysinefunctionalized with three or four polymeric subunits is within the scopeof the invention.

Specific embodiments according to the invention include:

and carbonates and active esters of these species, such as:

Other activating, or leaving groups, appropriate for activating linearPEGs of use in preparing the compounds set forth herein include, but arenot limited to the species:

PEG molecules that are activated with these and other species andmethods of making the activated PEGs are set forth in WO 04/083259.

Those of skill in the art will appreciate that one or more of the m-PEGarms of the branched polymer can be replaced by a PEG moiety with adifferent terminus, e.g., OH, COOH, NH₂, C₂-C₁₀-alkyl, etc. Moreover,the structures above are readily modified by inserting alkyl linkers (orremoving carbon atoms) between the α-carbon atom and the functionalgroup of the side chain. Thus, “homo” derivatives and higher homologues,as well as lower homologues are within the scope of cores for branchedPEGs of use in the present invention.

The branched PEG species set forth herein are readily prepared bymethods such as that set forth in the scheme below:

in which X^(a) is O or S and r is an integer from 1 to 5. The indices eand f are independently selected integers from 1 to 2500.

Thus, according to this scheme, a natural or unnatural amino acid iscontacted with an activated m-PEG derivative, in this case the tosylate,forming 1 by alkylating the side-chain heteroatom X^(a). Themono-functionalized m-PEG amino acid is submitted to N-acylationconditions with a reactive m-PEG derivative, thereby assembling branchedm-PEG 2. As one of skill will appreciate, the tosylate leaving group canbe replaced with any suitable leaving group, e.g., halogen, mesylate,triflate, etc. Similarly, the reactive carbonate utilized to acylate theamine can be replaced with an active ester, e.g., N-hydroxysuccinimide,etc., or the acid can be activated in situ using a dehydrating agentsuch as dicyclohexylcarbodiimide, carbonyldiimidazole, etc.

In an exemplary embodiment, the modifying group is a PEG moiety,however, any modifying group, e.g., water-soluble polymer,water-insoluble polymer, therapeutic moiety, etc., can be incorporatedin a glycosyl moiety through an appropriate linkage. The modified sugaris formed by enzymatic means, chemical means or a combination thereof,thereby producing a modified sugar. In an exemplary embodiment, thesugars are substituted with an active amine at any position that allowsfor the attachment of the modifying moiety, yet still allows the sugarto function as a substrate for an enzyme capable of coupling themodified sugar to the G-CSF peptide. In an exemplary embodiment, whengalactosamine is the modified sugar, the amine moiety is attached to thecarbon atom at the 6-position.

Water-soluble Polymer Modified Species

Water-soluble polymer modified nucleotide sugar species in which thesugar moiety is modified with a water-soluble polymer are of use in thepresent invention. An exemplary modified sugar nucleotide bears a sugargroup that is modified through an amine moiety on the sugar. Modifiedsugar nucleotides, e.g., saccharyl-amine derivatives of a sugarnucleotide, are also of use in the methods of the invention. Forexample, a saccharyl amine (without the modifying group) can beenzymatically conjugated to a peptide (or other species) and the freesaccharyl amine moiety subsequently conjugated to a desired modifyinggroup. Alternatively, the modified sugar nucleotide can function as asubstrate for an enzyme that transfers the modified sugar to a saccharylacceptor on a substrate, e.g., a peptide, glycopeptide, lipid, aglycone,glycolipid, etc.

In one embodiment in which the saccharide core is galactose or glucose,R⁵ is NHC(O)Y.

In an exemplary embodiment, the modified sugar is based upon a6-amino-N-acetyl-glycosyl moiety. As shown below forN-acetylgalactosamine, the 6-amino-sugar moiety is readily prepared bystandard methods.

In the scheme above, the index n represents an integer from 1 to 2500,preferably from 10 to 1500, and more preferably from 10 to 1200. Thesymbol “A” represents an activating group, e.g., a halo, a component ofan activated ester (e.g., a N-hydroxysuccinimide ester), a component ofa carbonate (e.g., p-nitrophenyl carbonate) and the like. Those of skillin the art will appreciate that other PEG-amide nucleotide sugars arereadily prepared by this and analogous methods.

In other exemplary embodiments, the amide moiety is replaced by a groupsuch as a urethane or a urea.

In still further embodiments, R¹ is a branched PEG, for example, one ofthose species set forth above. Illustrative compounds according to thisembodiment include:

in which X⁴ is a bond or O, and J is S or O.

Moreover, as discussed above, the present invention provides peptideconjugates that are formed using nucleotide sugars that are modifiedwith a water-soluble polymer, which is either straight-chain orbranched. For example, compounds having the formula shown below arewithin the scope of the present invention:

in which X⁴ is O or a bond, and J is S or O.

Similarly, the invention provides peptide conjugates that are formedusing nucleotide sugars of those modified sugar species in which thecarbon at the 6-position is modified:

in which X⁴ is a bond or O, J is S or O, and y is 0 or 1.

Also provided are conjugates of peptides and glycopeptides, lipids andglycolipids that include the compositions of the invention. For example,the invention provides conjugates having the following formulae:

wherein J s S or O.

Water-Insoluble Polymers

In another embodiment, analogous to those discussed above, the modifiedsugars include a water-insoluble polymer, rather than a water-solublepolymer. The conjugates of the invention may also include one or morewater-insoluble polymers. This embodiment of the invention isillustrated by the use of the conjugate as a vehicle with which todeliver a therapeutic peptide in a controlled manner. Polymeric drugdelivery systems are known in the art. See, for example, Dunn et al.,Eds. POLYMERIC DRUGS AND DRUG DELIVERY SYSTEMS, ACS Symposium SeriesVol. 469, American Chemical Society, Washington, D.C. 1991. Those ofskill in the art will appreciate that substantially any known drugdelivery system is applicable to the conjugates of the presentinvention.

Representative water-insoluble polymers include, but are not limited to,polyphosphazines, poly(vinyl alcohols), polyamides, polycarbonates,polyalkylenes, polyacrylamides, polyalkylene glycols, polyalkyleneoxides, polyalkylene terephthalates, polyvinyl ethers, polyvinyl esters,polyvinyl halides, polyvinylpyrrolidone, polyglycolides, polysiloxanes,polyurethanes, poly(methyl methacrylate), poly(ethyl methacrylate),poly(butyl methacrylate), poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate),poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropylacrylate), poly(isobutyl acrylate), poly(octadecyl acrylate)polyethylene, polypropylene, poly(ethylene glycol), poly(ethyleneoxide), poly (ethylene terephthalate), poly(vinyl acetate), polyvinylchloride, polystyrene, polyvinyl pyrrolidone, pluronics andpolyvinylphenol and copolymers thereof.

Synthetically modified natural polymers of use in conjugates of theinvention include, but are not limited to, alkyl celluloses,hydroxyalkyl celluloses, cellulose ethers, cellulose esters, andnitrocelluloses. Particularly preferred members of the broad classes ofsynthetically modified natural polymers include, but are not limited to,methyl cellulose, ethyl cellulose, hydroxypropyl cellulose,hydroxypropyl methyl cellulose, hydroxybutyl methyl cellulose, celluloseacetate, cellulose propionate, cellulose acetate butyrate, celluloseacetate phthalate, carboxymethyl cellulose, cellulose triacetate,cellulose sulfate sodium salt, and polymers of acrylic and methacrylicesters and alginic acid.

These and the other polymers discussed herein can be readily obtainedfrom commercial sources such as Sigma Chemical Co. (St. Louis, Mo.),Polysciences (Warrenton, Pa.), Aldrich (Milwaukee, Wis.), Fluka(Ronkonkoma, N.Y.), and BioRad (Richmond, Calif.), or else synthesizedfrom monomers obtained from these suppliers using standard techniques.

Representative biodegradable polymers of use in the conjugates of theinvention include, but are not limited to, polylactides, polyglycolidesand copolymers thereof, poly(ethylene terephthalate), poly(butyricacid), poly(valeric acid), poly(lactide-co-caprolactone),poly(lactide-co-glycolide), polyanhydrides, polyorthoesters, blends andcopolymers thereof. Of particular use are compositions that form gels,such as those including collagen, pluronics and the like.

The polymers of use in the invention include “hybrid” polymers thatinclude water-insoluble materials having within at least a portion oftheir structure, a bioresorbable molecule. An example of such a polymeris one that includes a water-insoluble copolymer, which has abioresorbable region, a hydrophilic region and a plurality ofcrosslinkable functional groups per polymer chain.

For purposes of the present invention, “water-insoluble materials”includes materials that are substantially insoluble in water orwater-containing environments. Thus, although certain regions orsegments of the copolymer may be hydrophilic or even water-soluble, thepolymer molecule, as a whole, does not to any substantial measuredissolve in water.

For purposes of the present invention, the term “bioresorbable molecule”includes a region that is capable of being metabolized or broken downand resorbed and/or eliminated through normal excretory routes by thebody. Such metabolites or break down products are preferablysubstantially non-toxic to the body.

The bioresorbable region may be either hydrophobic or hydrophilic, solong as the copolymer composition as a whole is not renderedwater-soluble. Thus, the bioresorbable region is selected based on thepreference that the polymer, as a whole, remains water-insoluble.Accordingly, the relative properties, i.e., the kinds of functionalgroups contained by, and the relative proportions of the bioresorbableregion, and the hydrophilic region are selected to ensure that usefulbioresorbable compositions remain water-insoluble.

Exemplary resorbable polymers include, for example, syntheticallyproduced resorbable block copolymers of poly(α-hydroxy-carboxylicacid)/poly(oxyalkylene, (see, Cohn et al., U.S. Pat. No. 4,826,945).These copolymers are not crosslinked and are water-soluble so that thebody can excrete the degraded block copolymer compositions. See, Youneset al., J. Biomed. Mater. Res. 21: 1301-1316 (1987); and Cohn et al., J.Biomed. Mater. Res. 22: 993-1009 (1988).

Presently preferred bioresorbable polymers include one or morecomponents selected from poly(esters), poly(hydroxy acids),poly(lactones), poly(amides), poly(ester-amides), poly(amino acids),poly(anhydrides), poly(orthoesters), poly(carbonates),poly(phosphazines), poly(phosphoesters), poly(thioesters),polysaccharides and mixtures thereof. More preferably still, thebioresorbable polymer includes a poly(hydroxy) acid component. Of thepoly(hydroxy) acids, polylactic acid, polyglycolic acid, polycaproicacid, polybutyric acid, polyvaleric acid and copolymers and mixturesthereof are preferred.

In addition to forming fragments that are absorbed in vivo(“bioresorbed”), preferred polymeric coatings for use in the methods ofthe invention can also form an excretable and/or metabolizable fragment.

Higher order copolymers can also be used in the present invention. Forexample, Casey et al., U.S. Pat. No. 4,438,253, which issued on Mar. 20,1984, discloses tri-block copolymers produced from thetransesterification of poly(glycolic acid) and an hydroxyl-endedpoly(alkylene glycol). Such compositions are disclosed for use asresorbable monofilament sutures. The flexibility of such compositions iscontrolled by the incorporation of an aromatic orthocarbonate, such astetra-p-tolyl orthocarbonate into the copolymer structure.

Other polymers based on lactic and/or glycolic acids can also beutilized. For example, Spinu, U.S. Pat. No. 5,202,413, which issued onApr. 13, 1993, discloses biodegradable multi-block copolymers havingsequentially ordered blocks of polylactide and/or polyglycolide producedby ring-opening polymerization of lactide and/or glycolide onto eitheran oligomeric diol or a diamine residue followed by chain extension witha di-functional compound, such as, a diisocyanate, diacylchloride ordichlorosilane.

Bioresorbable regions of coatings useful in the present invention can bedesigned to be hydrolytically and/or enzymatically cleavable. Forpurposes of the present invention, “hydrolytically cleavable” refers tothe susceptibility of the copolymer, especially the bioresorbableregion, to hydrolysis in water or a water-containing environment.Similarly, “enzymatically cleavable” as used herein refers to thesusceptibility of the copolymer, especially the bioresorbable region, tocleavage by endogenous or exogenous enzymes.

When placed within the body, the hydrophilic region can be processedinto excretable and/or metabolizable fragments. Thus, the hydrophilicregion can include, for example, polyethers, polyalkylene oxides,polyols, poly(vinyl pyrrolidine), poly(vinyl alcohol), poly(alkyloxazolines), polysaccharides, carbohydrates, peptides, proteins andcopolymers and mixtures thereof. Furthermore, the hydrophilic region canalso be, for example, a poly(alkylene) oxide. Such poly(alkylene) oxidescan include, for example, poly(ethylene) oxide, poly(propylene) oxideand mixtures and copolymers thereof.

Polymers that are components of hydrogels are also useful in the presentinvention. Hydrogels are polymeric materials that are capable ofabsorbing relatively large quantities of water. Examples of hydrogelforming compounds include, but are not limited to, polyacrylic acids,sodium carboxymethylcellulose, polyvinyl alcohol, polyvinyl pyrrolidine,gelatin, carrageenan and other polysaccharides,hydroxyethylenemethacrylic acid (HEMA), as well as derivatives thereof,and the like. Hydrogels can be produced that are stable, biodegradableand bioresorbable. Moreover, hydrogel compositions can include subunitsthat exhibit one or more of these properties.

Bio-compatible hydrogel compositions whose integrity can be controlledthrough crosslinking are known and are presently preferred for use inthe methods of the invention. For example, Hubbell et al., U.S. Pat.Nos. 5,410,016, which issued on Apr. 25, 1995 and 5,529,914, whichissued on Jun. 25, 1996, disclose water-soluble systems, which arecrosslinked block copolymers having a water-soluble central blocksegment sandwiched between two hydrolytically labile extensions. Suchcopolymers are further end-capped with photopolymerizable acrylatefunctionalities. When crosslinked, these systems become hydrogels. Thewater soluble central block of such copolymers can include poly(ethyleneglycol); whereas, the hydrolytically labile extensions can be apoly(α-hydroxy acid), such as polyglycolic acid or polylactic acid. See,Sawhney et al., Macromolecules 26: 581-587 (1993).

In another embodiment, the gel is a thermoreversible gel.Thermoreversible gels including components, such as pluronics, collagen,gelatin, hyalouronic acid, polysaccharides, polyurethane hydrogel,polyurethane-urea hydrogel and combinations thereof are presentlypreferred.

In yet another exemplary embodiment, the conjugate of the inventionincludes a component of a liposome. Liposomes can be prepared accordingto methods known to those skilled in the art, for example, as describedin Eppstein et al., U.S. Pat. No. 4,522,811, which issued on Jun. 11,1985. For example, liposome formulations may be prepared by dissolvingappropriate lipid(s) (such as stearoyl phosphatidyl ethanolamine,stearoyl phosphatidyl choline, arachadoyl phosphatidyl choline, andcholesterol) in an inorganic solvent that is then evaporated, leavingbehind a thin film of dried lipid on the surface of the container. Anaqueous solution of the active compound or its pharmaceuticallyacceptable salt is then introduced into the container. The container isthen swirled by hand to free lipid material from the sides of thecontainer and to disperse lipid aggregates, thereby forming theliposomal suspension.

The above-recited microparticles and methods of preparing themicroparticles are offered by way of example and they are not intendedto define the scope of microparticles of use in the present invention.It will be apparent to those of skill in the art that an array ofmicroparticles, fabricated by different methods, are of use in thepresent invention.

The structural formats discussed above in the context of thewater-soluble polymers, both straight-chain and branched are generallyapplicable with respect to the water-insoluble polymers as well. Thus,for example, the cysteine, serine, dilysine, and trilysine branchingcores can be functionalized with two water-insoluble polymer moieties.The methods used to produce these species are generally closelyanalogous to those used to produce the water-soluble polymers.

The in vivo half-life of therapeutic glycopeptides can also be enhancedwith PEG moieties such as polyethylene glycol (PEG). For example,chemical modification of proteins with PEG (PEGylation) increases theirmolecular size and decreases their surface- and functionalgroup-accessibility, each of which are dependent on the size of the PEGattached to the protein. This results in an improvement of plasmahalf-lives and in proteolytic-stability, and a decrease inimmunogenicity and hepatic uptake (Chaffee et al. J. Clin. Invest. 89:1643-1651 (1992); Pyatak et al. Res. Commun. Chem. Pathol Pharmacol. 29:113-127 (1980)). PEGylation of interleukin-2 has been reported toincrease its antitumor potency in vivo (Katre et al. Proc. Natl. Acad.Sci. USA. 84: 1487-1491 (1987)) and PEGylation of a F(ab′)2 derived fromthe monoclonal antibody A7 has improved its tumor localization (Kitamuraet al. Biochem. Biophys. Res. Commun. 28: 1387-1394 (1990)). Thus, inanother embodiment, the in vivo half-life of a peptide derivatized witha PEG moiety by a method of the invention is increased relevant to thein vivo half-life of the non-derivatized peptide.

The increase in peptide in vivo half-life is best expressed as a rangeof percent increase in this quantity. The lower end of the range ofpercent increase is about 40%, about 60%, about 80%, about 100%, about150% or about 200%. The upper end of the range is about 60%, about 80%,about 100%, about 150%, or more than about 250%.

Biomolecules

In another embodiment, the modified sugar bears a biomolecule. In stillfurther embodiments, the biomolecule is a functional protein, enzyme,antigen, antibody, peptide, nucleic acid (e.g., single nucleotides ornucleosides, oligonucleotides, polynucleotides and single- andhigher-stranded nucleic acids), lectin, receptor or a combinationthereof.

Preferred biomolecules are essentially non-fluorescent, or emit such aminimal amount of fluorescence that they are inappropriate for use as afluorescent marker in an assay. Moreover, it is generally preferred touse biomolecules that are not sugars. An exception to this preference isthe use of an otherwise naturally occurring sugar that is modified bycovalent attachment of another entity (e.g., PEG, biomolecule,therapeutic moiety, diagnostic moiety, etc.). In an exemplaryembodiment, a sugar moiety, which is a biomolecule, is conjugated to alinker arm and the sugar-linker arm cassette is subsequently conjugatedto a peptide via a method of the invention.

Biomolecules useful in practicing the present invention can be derivedfrom any source. The biomolecules can be isolated from natural sourcesor they can be produced by synthetic methods. Peptides can be naturalpeptides or mutated peptides. Mutations can be effected by chemicalmutagenesis, site-directed mutagenesis or other means of inducingmutations known to those of skill in the art. Peptides useful inpracticing the instant invention include, for example, enzymes,antigens, antibodies and receptors. Antibodies can be either polyclonalor monoclonal; either intact or fragments. The peptides are optionallythe products of a program of directed evolution

Both naturally derived and synthetic peptides and nucleic acids are ofuse in conjunction with the present invention; these molecules can beattached to a sugar residue component or a crosslinking agent by anyavailable reactive group. For example, peptides can be attached througha reactive amine, carboxyl, sulfhydryl, or hydroxyl group. The reactivegroup can reside at a peptide terminus or at a site internal to thepeptide chain. Nucleic acids can be attached through a reactive group ona base (e.g., exocyclic amine) or an available hydroxyl group on a sugarmoiety (e.g., 3′- or 5′-hydroxyl). The peptide and nucleic acid chainscan be further derivatized at one or more sites to allow for theattachment of appropriate reactive groups onto the chain. See, Chriseyet al. Nucleic Acids Res. 24: 3031-3039 (1996).

In a further embodiment, the biomolecule is selected to direct thepeptide modified by the methods of the invention to a specific tissue,thereby enhancing the delivery of the peptide to that tissue relative tothe amount of underivatized peptide that is delivered to the tissue. Ina still further embodiment, the amount of derivatized peptide deliveredto a specific tissue within a selected time period is enhanced byderivatization by at least about 20%, more preferably, at least about40%, and more preferably still, at least about 100%. Presently,preferred biomolecules for targeting applications include antibodies,hormones and ligands for cell-surface receptors.

In still a further exemplary embodiment, there is provided as conjugatewith biotin. Thus, for example, a selectively biotinylated peptide iselaborated by the attachment of an avidin or streptavidin moiety bearingone or more modifying groups.

Therapeutic Moieties

In another embodiment, the modified sugar includes a therapeutic moiety.Those of skill in the art will appreciate that there is overlap betweenthe category of therapeutic moieties and biomolecules; many biomoleculeshave therapeutic properties or potential.

The therapeutic moieties can be agents already accepted for clinical useor they can be drugs whose use is experimental, or whose activity ormechanism of action is under investigation. The therapeutic moieties canhave a proven action in a given disease state or can be onlyhypothesized to show desirable action in a given disease state. Inanother embodiment, the therapeutic moieties are compounds, which arebeing screened for their ability to interact with a tissue of choice.Therapeutic moieties, which are useful in practicing the instantinvention include drugs from a broad range of drug classes having avariety of pharmacological activities. Preferred therapeutic moietiesare essentially non-fluorescent, or emit such a minimal amount offluorescence that they are inappropriate for use as a fluorescent markerin an assay. Moreover, it is generally preferred to use therapeuticmoieties that are not sugars. An exception to this preference is the useof a sugar that is modified by covalent attachment of another entity,such as a PEG, biomolecule, therapeutic moiety, diagnostic moiety andthe like. In another exemplary embodiment, a therapeutic sugar moiety isconjugated to a linker arm and the sugar-linker arm cassette issubsequently conjugated to a peptide via a method of the invention.

Methods of conjugating therapeutic and diagnostic agents to variousother species are well known to those of skill in the art. See, forexample Hermanson, BIOCONJUGATE TECHNIQUES, Academic Press, San Diego,1996; and Dunn et al., Eds. POLYMERIC DRUGS AND DRUG DELIVERY SYSTEMS,ACS Symposium Series Vol. 469, American Chemical Society, Washington,D.C. 1991.

In an exemplary embodiment, the therapeutic moiety is attached to themodified sugar via a linkage that is cleaved under selected conditions.Exemplary conditions include, but are not limited to, a selected pH(e.g., stomach, intestine, endocytotic vacuole), the presence of anactive enzyme (e.g, esterase, reductase, oxidase), light, heat and thelike. Many cleavable groups are known in the art. See, for example, Junget al., Biochem. Biophys. Acta, 761: 152-162 (1983); Joshi et al., J.Biol. Chem., 265: 14518-14525 (1990); Zarling et al., J. Immunol., 124:913-920 (1980); Bouizar et al., Eur. J. Biochem., 155: 141-147 (1986);Park et al., J. Biol. Chem., 261: 205-210 (1986); Browning et al., J.Immunol., 143: 1859-1867 (1989).

Classes of useful therapeutic moieties include, for example,non-steroidal anti-inflammatory drugs (NSAIDS). The NSAIDS can, forexample, be selected from the following categories: (e.g., propionicacid derivatives, acetic acid derivatives, fenamic acid derivatives,biphenylcarboxylic acid derivatives and oxicams); steroidalanti-inflammatory drugs including hydrocortisone and the like;antihistaminic drugs (e.g., chlorpheniramine, triprolidine); antitussivedrugs (e.g., dextromethorphan, codeine, caramiphen and carbetapentane);antipruritic drugs (e.g., methdilazine and trimeprazine);anticholinergic drugs (e.g., scopolamine, atropine, homatropine,levodopa); anti-emetic and antinauseant drugs (e.g., cyclizine,meclizine, chlorpromazine, buclizine); anorexic drugs (e.g.,benzphetamine, phentermine, chlorphentermine, fenfluramine); centralstimulant drugs (e.g., amphetamine, methamphetamine, dextroamphetamineand methylphenidate); antiarrhythmic drugs (e.g., propanolol,procainamide, disopyramide, quinidine, encamide); β-adrenergic blockerdrugs (e.g., metoprolol, acebutolol, betaxolol, labetalol and timolol);cardiotonic drugs (e.g., milrinone, aminone and dobutamine);antihypertensive drugs (e.g., enalapril, clonidine, hydralazine,minoxidil, guanadrel, guanethidine); diuretic drugs (e.g., amiloride andhydrochlorothiazide); vasodilator drugs (e.g., diltiazem, amiodarone,isoxsuprine, nylidrin, tolazoline and verapamil); vasoconstrictor drugs(e.g., dihydroergotamine, ergotamine and methylsergide); antiulcer drugs(e.g., ranitidine and cimetidine); anesthetic drugs (e.g., lidocaine,bupivacaine, chloroprocaine, dibucaine); antidepressant drugs (e.g.,imipramine, desipramine, amitryptiline, nortryptiline); tranquilizer andsedative drugs (e.g., chlordiazepoxide, benacytyzine, benzquinamide,flurazepam, hydroxyzine, loxapine and promazine); antipsychotic drugs(e.g., chlorprothixene, fluphenazine, haloperidol, molindone,thioridazine and trifluoperazine); antimicrobial drugs (antibacterial,antifungal, antiprotozoal and antiviral drugs).

Antimicrobial drugs which are preferred for incorporation into thepresent composition include, for example, pharmaceutically acceptablesalts of β-lactam drugs, quinolone drugs, ciprofloxacin, norfloxacin,tetracycline, erythromycin, amikacin, triclosan, doxycycline,capreomycin, chlorhexidine, chlortetracycline, oxytetracycline,clindamycin, ethambutol, hexamidine isothionate, metronidazole,pentamidine, gentamycin, kanamycin, lineomycin, methacycline,methenamine, minocycline, neomycin, netilmycin, paromomycin,streptomycin, tobramycin, miconazole and amantadine.

Other drug moieties of use in practicing the present invention includeantineoplastic drugs (e.g., antiandrogens (e.g., leuprolide orflutamide), cytocidal agents (e.g., adriamycin, doxorubicin, taxol,cyclophosphamide, busulfan, cisplatin, β-2-interferon) anti-estrogens(e.g., tamoxifen), antimetabolites (e.g., fluorouracil, methotrexate,mercaptopurine, thioguanine). Also included within this class areradioisotope-based agents for both diagnosis and therapy, and conjugatedtoxins, such as ricin, geldanamycin, mytansin, CC-1065, theduocarmycins, Chlicheamycin and related structures and analoguesthereof.

The therapeutic moiety can also be a hormone (e.g., medroxyprogesterone,estradiol, leuprolide, megestrol, octreotide or somatostatin); musclerelaxant drugs (e.g., cinnamedrine, cyclobenzaprine, flavoxate,orphenadrine, papaverine, mebeverine, idaverine, ritodrine,diphenoxylate, dantrolene and azumolen); antispasmodic drugs;bone-active drugs (e.g., diphosphonate and phosphonoalkylphosphinatedrug compounds); endocrine modulating drugs (e.g., contraceptives (e.g.,ethinodiol, ethinyl estradiol, norethindrone, mestranol, desogestrel,medroxyprogesterone), modulators of diabetes (e.g., glyburide orchlorpropamide), anabolics, such as testolactone or stanozolol,androgens (e.g., methyltestosterone, testosterone or fluoxymesterone),antidiuretics (e.g., desmopressin) and calcitonins).

Also of use in the present invention are estrogens (e.g.,diethylstilbesterol), glucocorticoids (e.g., triamcinolone,betamethasone, etc.) and progestogens, such as norethindrone,ethynodiol, norethindrone, levonorgestrel; thyroid agents (e.g.,liothyronine or levothyroxine) or anti-thyroid agents (e.g.,methimazole); antihyperprolactinemic drugs (e.g., cabergoline); hormonesuppressors (e.g., danazol or goserelin), oxytocics (e.g.,methylergonovine or oxytocin) and prostaglandins, such as mioprostol,alprostadil or dinoprostone, can also be employed.

Other useful modifying groups include immunomodulating drugs (e.g.,antihistamines, mast cell stabilizers, such as lodoxamide and/orcromolyn, steroids (e.g., triamcinolone, beclomethazone, cortisone,dexamethasone, prednisolone, methylprednisolone, beclomethasone, orclobetasol), histamine H2 antagonists (e.g., famotidine, cimetidine,ranitidine), immunosuppressants (e.g., azathioprine, cyclosporin), etc.Groups with anti-inflammatory activity, such as sulindac, etodolac,ketoprofen and ketorolac, are also of use. Other drugs of use inconjunction with the present invention will be apparent to those ofskill in the art.

Preparation of Modified Sugars

In general, the sugar moiety and the modifying group are linked togetherthrough the use of reactive groups, which are typically transformed bythe linking process into a new organic functional group or unreactivespecies. The sugar reactive functional group(s), is located at anyposition on the sugar moiety. Reactive groups and classes of reactionsuseful in practicing the present invention are generally those that arewell known in the art of bioconjugate chemistry. Currently favoredclasses of reactions available with reactive sugar moieties are those,which proceed under relatively mild conditions. These include, but arenot limited to nucleophilic substitutions (e.g., reactions of amines andalcohols with acyl halides, active esters), electrophilic substitutions(e.g., enamine reactions) and additions to carbon-carbon andcarbon-heteroatom multiple bonds (e.g., Michael reaction, Diels-Alderaddition). These and other useful reactions are discussed in, forexample, March, ADVANCED ORGANIC CHEMISTRY, 3rd Ed., John Wiley & Sons,New York, 1985; Hermanson, BIOCONJUGATE TECHNIQUES, Academic Press, SanDiego, 1996; and Feeney et al., MODIFICATION OF PROTEINS; Advances inChemistry Series, Vol. 198, American Chemical Society, Washington, D.C.,1982.

Useful reactive functional groups pendent from a sugar nucleus ormodifying group include, but are not limited to:

-   -   (a) carboxyl groups and various derivatives thereof including,        but not limited to, N-hydroxysuccinimide esters,        N-hydroxybenztriazole esters, acid halides, acyl imidazoles,        thioesters, p-nitrophenyl esters, alkyl, alkenyl, alkynyl and        aromatic esters;    -   (b) hydroxyl groups, which can be converted to, e.g., esters,        ethers, aldehydes, etc.    -   (c) haloalkyl groups, wherein the halide can be later displaced        with a nucleophilic group such as, for example, an amine, a        carboxylate anion, thiol anion, carbanion, or an alkoxide ion,        thereby resulting in the covalent attachment of a new group at        the functional group of the halogen atom;    -   (d) dienophile groups, which are capable of participating in        Diels-Alder reactions such as, for example, maleimido groups;    -   (e) aldehyde or ketone groups, such that subsequent        derivatization is possible via formation of carbonyl derivatives        such as, for example, imines, hydrazones, semicarbazones or        oximes, or via such mechanisms as Grignard addition or        alkyllithium addition;    -   (f) sulfonyl halide groups for subsequent reaction with amines,        for example, to form sulfonamides;    -   (g) thiol groups, which can be, for example, converted to        disulfides or reacted with acyl halides;    -   (h) amine or sulfhydryl groups, which can be, for example,        acylated, alkylated or oxidized;    -   (i) alkenes, which can undergo, for example, cycloadditions,        acylation, Michael addition, etc; and    -   (j) epoxides, which can react with, for example, amines and        hydroxyl compounds.

The reactive functional groups can be chosen such that they do notparticipate in, or interfere with, the reactions necessary to assemblethe reactive sugar nucleus or modifying group. Alternatively, a reactivefunctional group can be protected from participating in the reaction bythe presence of a protecting group. Those of skill in the art understandhow to protect a particular functional group such that it does notinterfere with a chosen set of reaction conditions. For examples ofuseful protecting groups, see, for example, Greene et al., PROTECTIVEGROUPS IN ORGANIC SYNTHESIS, John Wiley & Sons, New York, 1991.

In the discussion that follows, a number of specific examples ofmodified sugars that are useful in practicing the present invention areset forth. In the exemplary embodiments, a sialic acid derivative isutilized as the sugar nucleus to which the modifying group is attached.The focus of the discussion on sialic acid derivatives is for clarity ofillustration only and should not be construed to limit the scope of theinvention. Those of skill in the art will appreciate that a variety ofother sugar moieties can be activated and derivatized in a manneranalogous to that set forth using sialic acid as an example. Forexample, numerous methods are available for modifying galactose,glucose, N-acetylgalactosamine and fucose to name a few sugarsubstrates, which are readily modified by art recognized methods. See,for example, Elhalabi et al., Curr. Med. Chem. 6: 93 (1999); and Schaferet al., J. Org. Chem. 65: 24 (2000)).

In an exemplary embodiment, the peptide that is modified by a method ofthe invention is a glycopeptide that is produced in prokaryotic cells(e.g., E. coli), eukaryotic cells including yeast and mammalian cells(e.g., CHO cells), or in a transgenic animal and thus contains N- and/orO-linked oligosaccharide chains, which are incompletely sialylated. Theoligosaccharide chains of the glycopeptide lacking a sialic acid andcontaining a terminal galactose residue can be glyco-PEG-ylated,glyco-PPG-ylated or otherwise modified with a modified sialic acid.

In Scheme 4, the amino glycoside 1, is treated with the active ester ofa protected amino acid (e.g., glycine) derivative, converting the sugaramine residue into the corresponding protected amino acid amide adduct.The adduct is treated with an aldolase to form α-hydroxy carboxylate 2.Compound 2 is converted to the corresponding CMP derivative by theaction of CMP-SA synthetase, followed by catalytic hydrogenation of theCMP derivative to produce compound 3. The amine introduced via formationof the glycine adduct is utilized as a locus of PEG or PPG attachment byreacting compound 3 with an activated (m-) PEG or (m-) PPG derivative(e.g., PEG-C(O)NHS, PPG-C(O)NHS), producing 4 or 5, respectively.

Table 2 sets forth representative examples of sugar monophosphates thatare derivatized with a PEG or PPG moiety. Certain of the compounds ofTable 2 are prepared by the method of Scheme 4. Other derivatives areprepared by art-recognized methods. See, for example, Keppler et al.,Glycobiology 11: 11R (2001); and Charter et al., Glycobiology 10: 1049(2000)). Other amine reactive PEG and PPG analogues are commerciallyavailable, or they can be prepared by methods readily accessible tothose of skill in the art.

TABLE 2

The modified sugar phosphates of use in practicing the present inventioncan be substituted in other positions as well as those set forth above.Presently preferred substitutions of sialic acid are set forth inFormula I:

in which X is a linking group, which is preferably selected from —O—,—N(H)—, —S, CH₂—, and —N(R)₂, in which each R is a member independentlyselected from R¹-R⁵. The symbols Y, Z, A and B each represent a groupthat is selected from the group set forth above for the identity of X,X, Y, Z, A and B are each independently selected and, therefore, theycan be the same or different. The symbols R¹, R², R³, R⁴ and R⁵represent H, a water-soluble polymer, therapeutic moiety, biomolecule orother moiety. Alternatively, these symbols represent a linker that isbound to a water-soluble polymer, therapeutic moiety, biomolecule orother moiety.

Exemplary moieties attached to the conjugates disclosed herein include,but are not limited to, PEG derivatives (e.g., alkyl-PEG, acyl-PEG,acyl-alkyl-PEG, alkyl-acyl-PEG carbamoyl-PEG, aryl-PEG), PPG derivatives(e.g., alkyl-PPG, acyl-PPG, acyl-alkyl-PPG, alkyl-acyl-PPGcarbamoyl-PPG, aryl-PPG), therapeutic moieties, diagnostic moieties,mannose-6-phosphate, heparin, heparan, SLex, mannose,mannose-6-phosphate, Sialyl Lewis X, FGF, VFGF, proteins, chondroitin,keratan, dermatan, albumin, integrins, antennary oligosaccharides,peptides and the like. Methods of conjugating the various modifyinggroups to a saccharide moiety are readily accessible to those of skillin the art (POLY (ETHYLENE GLYCOL CHEMISTRY: BIOTECHNICAL AND BIOMEDICALAPPLICATIONS, J. Milton Harris, Ed., Plenum Pub. Corp., 1992; POLY(ETHYLENE GLYCOL) CHEMICAL AND BIOLOGICAL APPLICATIONS, J. MiltonHarris, Ed., ACS Symposium Series No. 680, American Chemical Society,1997; Hermanson, BIOCONJUGATE TECHNIQUES, Academic Press, San Diego,1996; and Dunn et al., Eds. POLYMERIC DRUGS AND DRUG DELIVERYSYSTEMS,ACS Symposium Series Vol. 469, American Chemical Society, Washington,D.C. 1991).

Cross-Linking Groups

Preparation of the Modified Sugar for Use in the Methods of the PresentInvention includes attachment of a modifying group to a sugar residueand forming a stable adduct, which is a substrate for aglycosyltransferase. The sugar and modifying group can be coupled by azero- or higher-order cross-linking agent. Exemplary bifunctionalcompounds which can be used for attaching modifying groups tocarbohydrate moieties include, but are not limited to, bifunctionalpoly(ethyleneglycols), polyamides, polyethers, polyesters and the like.General approaches for linking carbohydrates to other molecules areknown in the literature. See, for example, Lee et al., Biochemistry 28:1856 (1989); Bhatia et al., Anal. Biochem. 178: 408 (1989); Janda etal., J. Am. Chem. Soc. 112: 8886 (1990) and Bednarski et al., WO92/18135. In the discussion that follows, the reactive groups aretreated as benign on the sugar moiety of the nascent modified sugar. Thefocus of the discussion is for clarity of illustration. Those of skillin the art will appreciate that the discussion is relevant to reactivegroups on the modifying group as well.

An exemplary strategy involves incorporation of a protected sulfhydrylonto the sugar using the heterobifunctional crosslinker SPDP(n-succinimidyl-3-(2-pyridyldithio)propionate and then deprotecting thesulfhydryl for formation of a disulfide bond with another sulfhydryl onthe modifying group.

If SPDP detrimentally affects the ability of the modified sugar to actas a glycosyltransferase substrate, one of an array of othercrosslinkers such as 2-iminothiolane or N-succinimidylS-acetylthioacetate (SATA) is used to form a disulfide bond.2-iminothiolane reacts with primary amines, instantly incorporating anunprotected sulfhydryl onto the amine-containing molecule. SATA alsoreacts with primary amines, but incorporates a protected sulfhydryl,which is later deacetaylated using hydroxylamine to produce a freesulfhydryl. In each case, the incorporated sulfhydryl is free to reactwith other sulfhydryls or protected sulfhydryl, like SPDP, forming therequired disulfide bond.

The above-described strategy is exemplary, and not limiting, of linkersof use in the invention. Other crosslinkers are available that can beused in different strategies for crosslinking the modifying group to thepeptide. For example, TPCH(S-(2-thiopyridyl)-L-cysteine hydrazide andTPMPH ((S-(2-thiopyridyl) mercapto-propionohydrazide) react withcarbohydrate moieties that have been previously oxidized by mildperiodate treatment, thus forming a hydrazone bond between the hydrazideportion of the crosslinker and the periodate generated aldehydes. TPCHand TPMPH introduce a 2-pyridylthione protected sulfhydryl group ontothe sugar, which can be deprotected with DTT and then subsequently usedfor conjugation, such as forming disulfide bonds between components.

If disulfide bonding is found unsuitable for producing stable modifiedsugars, other crosslinkers may be used that incorporate more stablebonds between components. The heterobifunctional crosslinkers GMBS(N-gama-malimidobutyryloxy)succinimide) and SMCC (succinimidyl4-(N-maleimido-methyl)cyclohexane) react with primary amines, thusintroducing a maleimide group onto the component. The maleimide groupcan subsequently react with sulfhydryls on the other component, whichcan be introduced by previously mentioned crosslinkers, thus forming astable thioether bond between the components. If steric hindrancebetween components interferes with either component's activity or theability of the modified sugar to act as a glycosyltransferase substrate,crosslinkers can be used which introduce long spacer arms betweencomponents and include derivatives of some of the previously mentionedcrosslinkers (i.e., SPDP). Thus, there is an abundance of suitablecrosslinkers, which are useful; each of which is selected depending onthe effects it has on optimal peptide conjugate and modified sugarproduction.

A variety of reagents are used to modify the components of the modifiedsugar with intramolecular chemical crosslinks (for reviews ofcrosslinking reagents and crosslinking procedures see: Wold, F., Meth.Enzymol. 25: 623-651, 1972; Weetall, H. H., and Cooney, D. A., In:ENZYMES AS DRUGS. (Holcenberg, and Roberts, eds.) pp. 395-442, Wiley,New York, 1981; Ji, T. H., Meth. Enzymol. 91: 580-609, 1983; Mattson etal., Mol. Biol. Rep. 17: 167-183, 1993, all of which are incorporatedherein by reference). Preferred crosslinking reagents are derived fromvarious zero-length, homo-bifunctional, and hetero-bifunctionalcrosslinking reagents. Zero-length crosslinking reagents include directconjugation of two intrinsic chemical groups with no introduction ofextrinsic material. Agents that catalyze formation of a disulfide bondbelong to this category. Another example is reagents that inducecondensation of a carboxyl and a primary amino group to form an amidebond such as carbodiimides, ethylchloroformate, Woodward's reagent K(2-ethyl-5-phenylisoxazolium-3′-sulfonate), and carbonyldiimidazole. Inaddition to these chemical reagents, the enzyme transglutaminase(glutamyl-peptide γ-glutamyltransferase; EC 2.3.2.13) may be used aszero-length crosslinking reagent. This enzyme catalyzes acyl transferreactions at carboxamide groups of protein-bound glutaminyl residues,usually with a primary amino group as substrate. Preferred homo- andhetero-bifunctional reagents contain two identical or two dissimilarsites, respectively, which may be reactive for amino, sulfhydryl,guanidino, indole, or nonspecific groups.

i. Preferred Specific Sites in Crosslinking Reagents

1. Amino-Reactive Groups

In one embodiment, the sites on the cross-linker are amino-reactivegroups. Useful non-limiting examples of amino-reactive groups includeN-hydroxysuccinimide (NHS) esters, imidoesters, isocyanates,acylhalides, arylazides, p-nitrophenyl esters, aldehydes, and sulfonylchlorides.

NHS esters react preferentially with the primary (including aromatic)amino groups of a modified sugar component. The imidazole groups ofhistidines are known to compete with primary amines for reaction, butthe reaction products are unstable and readily hydrolyzed. The reactioninvolves the nucleophilic attack of an amine on the acid carboxyl of anNHS ester to form an amide, releasing the N-hydroxysuccinimide. Thus,the positive charge of the original amino group is lost.

Imidoesters are the most specific acylating reagents for reaction withthe amine groups of the modified sugar components. At a pH between 7 and10, imidoesters react only with primary amines. Primary amines attackimidates nucleophilically to produce an intermediate that breaks down toamidine at high pH or to a new imidate at low pH. The new imidate canreact with another primary amine, thus crosslinking two amino groups, acase of a putatively monofunctional imidate reacting bifunctionally. Theprincipal product of reaction with primary amines is an amidine that isa stronger base than the original amine. The positive charge of theoriginal amino group is therefore retained.

Isocyanates (and isothiocyanates) react with the primary amines of themodified sugar components to form stable bonds. Their reactions withsulflhydryl, imidazole, and tyrosyl groups give relatively unstableproducts.

Acylazides are also used as amino-specific reagents in whichnucleophilic amines of the affinity component attack acidic carboxylgroups under slightly alkaline conditions, e.g. pH 8.5.

Arylhalides such as 1,5-difluoro-2,4-dinitrobenzene react preferentiallywith the amino groups and tyrosine phenolic groups of modified sugarcomponents, but also with sulfhydryl and imidazole groups.

p-Nitrophenyl esters of mono- and dicarboxylic acids are also usefulamino-reactive groups. Although the reagent specificity is not veryhigh, α- and ε-amino groups appear to react most rapidly.

Aldehydes such as glutaraldehyde react with primary amines of modifiedsugar. Although unstable Schiff bases are formed upon reaction of theamino groups with the aldehydes of the aldehydes, glutaraldehyde iscapable of modifying the modified sugar with stable crosslinks. At pH6-8, the pH of typical crosslinking conditions, the cyclic polymersundergo a dehydration to form α-βunsaturated aldehyde polymers. Schiffbases, however, are stable, when conjugated to another double bond. Theresonant interaction of both double bonds prevents hydrolysis of theSchiff linkage. Furthermore, amines at high local concentrations canattack the ethylenic double bond to form a stable Michael additionproduct.

Aromatic sulfonyl chlorides react with a variety of sites of themodified sugar components, but reaction with the amino groups is themost important, resulting in a stable sulfonamide linkage.

2. Sulfhydryl-Reactive Groups

In another embodiment, the sites are sulfhydryl-reactive groups. Useful,non-limiting examples of sulfhydryl-reactive groups include maleimides,alkyl halides, pyridyl disulfides, and thiophthalimides.

Maleimides react preferentially with the sulfhydryl group of themodified sugar components to form stable thioether bonds. They alsoreact at a much slower rate with primary amino groups and the imidazolegroups of histidines. However, at pH 7 the maleimide group can beconsidered a sulfhydryl-specific group, since at this pH the reactionrate of simple thiols is 1000-fold greater than that of thecorresponding amine.

Alkyl halides react with sulfhydryl groups, sulfides, imidazoles, andamino groups. At neutral to slightly alkaline pH, however, alkyl halidesreact primarily with sulfhydryl groups to form stable thioether bonds.At higher pH, reaction with amino groups is favored.

Pyridyl disulfides react with free sulfhydryls via disulfide exchange togive mixed disulfides. As a result, pyridyl disulfides are the mostspecific sulfhydryl-reactive groups.

Thiophthalimides react with free sulfhydryl groups to form disulfides.

3. Carboxyl-Reactive Residue

In another embodiment, carbodiimides soluble in both water and organicsolvent, are used as carboxyl-reactive reagents. These compounds reactwith free carboxyl groups forming a pseudourea that can then couple toavailable amines yielding an amide linkage teach how to modify acarboxyl group with carbodiimde (Yamada et al., Biochemistry 20:4836-4842, 1981).

ii. Preferred Nonspecific Sites in Crosslinking Reagents

In addition to the use of site-specific reactive moieties, the presentinvention contemplates the use of non-specific reactive groups to linkthe sugar to the modifying group.

Exemplary non-specific cross-linkers include photoactivatable groups,completely inert in the dark, which are converted to reactive speciesupon absorption of a photon of appropriate energy. In one embodiment,photoactivatable groups are selected from precursors of nitrenesgenerated upon heating or photolysis of azides. Electron-deficientnitrenes are extremely reactive and can react with a variety of chemicalbonds including N—H, O—H, C—H, and C═C. Although three types of azides(aryl, alkyl, and acyl derivatives) may be employed, arylazides arepresently. The reactivity of arylazides upon photolysis is better withN—H and O—H than C—H bonds. Electron-deficient arylnitrenes rapidlyring-expand to form dehydroazepines, which tend to react withnucleophiles, rather than form C—H insertion products. The reactivity ofarylazides can be increased by the presence of electron-withdrawingsubstituents such as nitro or hydroxyl groups in the ring. Suchsubstituents push the absorption maximum of arylazides to longerwavelength. Unsubstituted arylazides have an absorption maximum in therange of 260-280 nm, while hydroxy and nitroarylazides absorbsignificant light beyond 305 nm. Therefore, hydroxy and nitroarylazidesare most preferable since they allow to employ less harmful photolysisconditions for the affinity component than unsubstituted arylazides.

In another preferred embodiment, photoactivatable groups are selectedfrom fluorinated arylazides. The photolysis products of fluorinatedarylazides are arylnitrenes, all of which undergo the characteristicreactions of this group, including C—H bond insertion, with highefficiency (Keana et al., J. Org. Chem. 55: 3640-3647, 1990).

In another embodiment, photoactivatable groups are selected frombenzophenone residues. Benzophenone reagents generally give highercrosslinking yields than arylazide reagents.

In another embodiment, photoactivatable groups are selected from diazocompounds, which form an electron-deficient carbene upon photolysis.These carbenes undergo a variety of reactions including insertion intoC—H bonds, addition to double bonds (including aromatic systems),hydrogen attraction and coordination to nucleophilic centers to givecarbon ions.

In still another embodiment, photoactivatable groups are selected fromdiazopyruvates. For example, the p-nitrophenyl ester of p-nitrophenyldiazopyruvate reacts with aliphatic amines to give diazopyruvic acidamides that undergo ultraviolet photolysis to form aldehydes. Thephotolyzed diazopyruvate-modified affinity component will react likeformaldehyde or glutaraldehyde forming crosslinks.

iii. Homobifunctional Reagents1. Homobifunctional Crosslinkers Reactive with Primary Amines

Synthesis, properties, and applications of amine-reactive cross-linkersare commercially described in the literature (for reviews ofcrosslinking procedures and reagents, see above). Many reagents areavailable (e.g., Pierce Chemical Company, Rockford, Ill.; Sigma ChemicalCompany, St. Louis, Mo.; Molecular Probes, Inc., Eugene, Oreg.).

Preferred, non-limiting examples of homobifunctional NHS esters includedisuccinimidyl glutarate (DSG), disuccinimidyl suberate (DSS),bis(sulfosuccinimidyl) suberate (BS), disuccinimidyl tartarate (DST),disulfosuccinimidyl tartarate (sulfo-DST),bis-2-(succinimidooxycarbonyloxy)ethylsulfone (BSOCOES),bis-2-(sulfosuccinimidooxy-carbonyloxy)ethylsulfone (sulfo-BSOCOES),ethylene glycolbis(succinimidylsuccinate) (EGS), ethyleneglycolbis(sulfosuccinimidylsuccinate) (sulfo-EGS),dithiobis(succinimidyl-propionate (DSP), anddithiobis(sulfosuccinimidylpropionate (sulfo-DSP). Preferred,non-limiting examples of homobifunctional imidoesters include dimethylmalonimidate (DMM), dimethyl succinimidate (DMSC), dimethyl adipimidate(DMA), dimethyl pimelimidate (DMP), dimethyl suberimidate (DMS),dimethyl-3,3′-oxydipropionimidate (DODP),dimethyl-3,3′-(methylenedioxy)dipropionimidate (DMDP),dimethyl-,3′-(dimethylenedioxy)dipropionimidate (DDDP),dimethyl-3,3′-(tetramethylenedioxy)-dipropionimidate (DTDP), anddimethyl-3,3′-dithiobispropionimidate (DTBP).

Preferred, non-limiting examples of homobifunctional isothiocyanatesinclude: p-phenylenediisothiocyanate (DITC), and4,4′-diisothiocyano-2,2′-disulfonic acid stilbene (DIDS).

Preferred, non-limiting examples of homobifunctional isocyanates includexylene-diisocyanate, toluene-2,4-diisocyanate,toluene-2-isocyanate-4-isothiocyanate,3-methoxydiphenylmethane-4,4′-diisocyanate,2,2′-dicarboxy-4,4′-azophenyldiisocyanate, andhexamethylenediisocyanate.

Preferred, non-limiting examples of homobifunctional arylhalides include1,5-difluoro-2,4-dinitrobenzene (DFDNB), and4,4′-difluoro-3,3′-dinitrophenyl-sulfone.

Preferred, non-limiting examples of homobifunctional aliphatic aldehydereagents include glyoxal, malondialdehyde, and glutaraldehyde.

Preferred, non-limiting examples of homobifunctional acylating reagentsinclude nitrophenyl esters of dicarboxylic acids.

Preferred, non-limiting examples of homobifunctional aromatic sulfonylchlorides include phenol-2,4-disulfonyl chloride, andα-naphthol-2,4-disulfonyl chloride.

Preferred, non-limiting examples of additional amino-reactivehomobifunctional reagents include erythritolbiscarbonate which reactswith amines to give biscarbamates.

2. Homobifunctional Crosslinkers Reactive with Free Sulfhydryl Groups

Synthesis, properties, and applications of such reagents are describedin the literature (for reviews of crosslinking procedures and reagents,see above). Many of the reagents are commercially available (e.g.,Pierce Chemical Company, Rockford, Ill.; Sigma Chemical Company, St.Louis, Mo.; Molecular Probes, Inc., Eugene, Oreg.).

Preferred, non-limiting examples of homobifunctional maleimides includebismaleimidohexane (BMH), N,N′-(1,3-phenylene)bismaleimide,N,N′-(1,2-phenylene)bismaleimide, azophenyldimaleimide, andbis(N-maleimidomethyl)ether.

Preferred, non-limiting examples of homobifunctional pyridyl disulfidesinclude 1,4-di-3′-(2′-pyridyldithio)propionamidobutane (DPDPB).

Preferred, non-limiting examples of homobifunctional alkyl halidesinclude 2,2′-dicarboxy-4,4′-diiodoacetamidoazobenzene,α,α′-diiodo-p-xylenesulfonic acid, α, α′-dibromo-p-xylenesulfonic acid,N,N′-bis(b-bromoethyl)benzylamine, N,N′-di(bromoacetyl)phenylthydrazine,and 1,2-di(bromoacetyl)amino-3-phenylpropane.

3. Homobifunctional Photoactivatable Crosslinkers

Synthesis, properties, and applications of such reagents are describedin the literature (for reviews of crosslinking procedures and reagents,see above). Some of the reagents are commercially available (e.g.,Pierce Chemical Company, Rockford, Ill.; Sigma Chemical Company, St.Louis, Mo.; Molecular Probes, Inc., Eugene, Oreg.).

Preferred, non-limiting examples of homobifunctional photoactivatablecrosslinker include bis-β-(4-azidosalicylamido)ethyldisulfide (BASED),di-N-(2-nitro-4-azidophenyl)-cystamine-S,S-dioxide (DNCO), and4,4′-dithiobisphenylazide.

iv. HeteroBifunctional Reagents1. Amino-Reactive HeteroBifunctional Reagents with a Pyridyl DisulfideMoiety

Synthesis, properties, and applications of such reagents are describedin the literature (for reviews of crosslinking procedures and reagents,see above). Many of the reagents are commercially available (e.g.,Pierce Chemical Company, Rockford, Ill.; Sigma Chemical Company, St.Louis, Mo.; Molecular Probes, Inc., Eugene, Oreg.).

Preferred, non-limiting examples of hetero-bifunctional reagents with apyridyl disulfide moiety and an amino-reactive NHS ester includeN-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl6-3-(2-pyridyldithio)propionamidohexanoate (LC-SPDP), sulfosuccinimidyl6-3-(2-pyridyldithio)propionamidohexanoate (sulfo-LCSPDP),4-succinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio)toluene (SMPT),and sulfosuccinimidyl 6-α-methyl-α-(2-pyridyldithio)toluamidohexanoate(sulfo-LC-SMPT).

2. Amino-Reactive HeteroBifunctional Reagents with a Maleimide Moiety

Synthesis, properties, and applications of such reagents are describedin the literature. Preferred, non-limiting examples ofhetero-bifunctional reagents with a maleimide moiety and anamino-reactive NHS ester include succinimidyl maleimidylacetate (AMAS),succinimidyl 3-maleimidylpropionate (BMPS),N-γ-maleimidobutyryloxysuccinimide ester(GMBS)N-γ-maleimidobutyryloxysulfo succinimide ester (sulfo-GMBS)succinimidyl 6-maleimidylhexanoate (EMCS), succinimidyl3-maleimidylbenzoate (SMB), m-maleimidobenzoyl-N-hydroxysuccinimideester (MBS), m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester(sulfo-MBS), succinimidyl4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC),sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate(sulfo-SMCC), succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB), andsulfosuccinimidyl 4-(p-maleimidophenyl)butyrate (sulfo-SMPB).

3. Amino-Reactive HeteroBifunctional Reagents with an Alkyl HalideMoiety

Synthesis, properties, and applications of such reagents are describedin the literature Preferred, non-limiting examples ofhetero-bifunctional reagents with an alkyl halide moiety and anamino-reactive NHS ester includeN-succinimidyl-(4-iodoacetyl)aminobenzoate (SIAB),sulfosuccinimidyl-(4-iodoacetyl)aminobenzoate (sulfo-SIAB),succinimidyl-6-(iodoacetyl)aminohexanoate (SIAX),succinimidyl-6-(6-((iodoacetyl)-amino)hexanoylamino)hexanoate (SIAXX),succinimidyl-6-(((4-(iodoacetyl)-amino)-methyl)-cyclohexane-1-carbonyl)aminohexanoate(SIACX), andsuccinimidyl-4((iodoacetyl)-amino)methylcyclohexane-1-carboxylate(SIAC).

An example of a hetero-bifunctional reagent with an amino-reactive NHSester and an alkyl dihalide moiety is N-hydroxysuccinimidyl2,3-dibromopropionate (SDBP). SDBP introduces intramolecular crosslinksto the affinity component by conjugating its amino groups. Thereactivity of the dibromopropionyl moiety towards primary amine groupsis controlled by the reaction temperature (McKenzie et al., ProteinChem. 7: 581-592 (1988)).

Preferred, non-limiting examples of hetero-bifunctional reagents with analkyl halide moiety and an amino-reactive p-nitrophenyl ester moietyinclude p-nitrophenyl iodoacetate (NPIA).

Other cross-linking agents are known to those of skill in the art. See,for example, Pomato et al., U.S. Pat. No. 5,965,106. It is within theabilities of one of skill in the art to choose an appropriatecross-linking agent for a particular application.

v. Cleavable Linker Groups

In yet a further embodiment, the linker group is provided with a groupthat can be cleaved to release the modifying group from the sugarresidue. Many cleaveable groups are known in the art. See, for example,Jung et al., Biochem. Biophys. Acta 761: 152-162 (1983); Joshi et al.,J. Biol. Chem. 265: 14518-14525 (1990); Zarling et al., J. Immunol. 124:913-920 (1980); Bouizar et al., Eur. J. Biochem. 155: 141-147 (1986);Park et al., J. Biol. Chem. 261: 205-210 (1986); Browning et al., J.Immunol. 143: 1859-1867 (1989). Moreover a broad range of cleavable,bifunctional (both homo- and hetero-bifunctional) linker groups iscommercially available from suppliers such as Pierce.

Exemplary cleaveable moieties can be cleaved using light, heat orreagents such as thiols, hydroxylamine, bases, periodate and the like.Moreover, certain preferred groups are cleaved in vivo in response tobeing endocytized (e.g., cis-aconityl; see, Shen et al., Biochem.Biophys. Res. Commun. 102: 1048 (1991)). Preferred cleaveable groupscomprise a cleaveable moiety which is a member selected from the groupconsisting of disulfide, ester, imide, carbonate, nitrobenzyl, phenacyland benzoin groups.

Conjugation of Modified Sugars to Peptides

The modified sugars are conjugated to a glycosylated or non-glycosylatedpeptide using an appropriate enzyme to mediate the conjugation.Preferably, the concentrations of the modified donor sugar(s), enzyme(s)and acceptor peptide(s) are selected such that glycosylation proceedsuntil the acceptor is consumed. The considerations discussed below,while set forth in the context of a sialyltransferase, are generallyapplicable to other glycosyltransferase reactions.

A number of methods of using glycosyltransferases to synthesize desiredoligosaccharide structures are known and are generally applicable to theinstant invention. Exemplary methods are described, for instance, WO96/32491, Ito et al., Pure Appl. Chem. 65: 753 (1993), and U.S. Pat.Nos. 5,352,670, 5,374,541, and 5,545,553.

The present invention is practiced using a single glycosyltransferase ora combination of glycosyltransferases. For example, one can use acombination of a sialyltransferase and a galactosyltransferase. In thoseembodiments using more than one enzyme, the enzymes and substrates arepreferably combined in an initial reaction mixture, or the enzymes andreagents for a second enzymatic reaction are added to the reactionmedium once the first enzymatic reaction is complete or nearly complete.By conducting two enzymatic reactions in sequence in a single vessel,overall yields are improved over procedures in which an intermediatespecies is isolated. Moreover, cleanup and disposal of extra solventsand by-products is reduced.

In another embodiment, each of the first and second enzyme is aglycosyltransferase. In another embodiment, one enzyme is anendoglycosidase. In an additional embodiment, more than two enzymes areused to assemble the modified glycoprotein of the invention. The enzymesare used to alter a saccharide structure on the peptide at any pointeither before or after the addition of the modified sugar to thepeptide.

The O-linked glycosyl moieties of the conjugates of the invention aregenerally originate with a GalNAc moiety that is attached to thepeptide. Any member of the family of GalNAc transferases can be used tobind a GalNAc moiety to the peptide (Hassan H, Bennett EP, Mandel U,Hollingsworth Mass., and Clausen H (2000). Control of Mucin-TypeO-Glycosylation: O-Glycan Occupancy is Directed by SubstrateSpecificities of Polypeptide GalNAc-Transferases. (Eds. Ernst, Hart, andSinay). Wiley-VCH chapter “Carbohydrates in Chemistry and Biology—aComprehension Handbook”, 273-292). The GalNAc moiety itself can be theintact glycosyl linker. Alternatively, the saccharyl residue is builtout using one more enzyme and one or more appropriate glycosyl substratefor the enzyme, the modified sugar being added to the built out glycosylmoiety.

In another embodiment, the method makes use of one or more exo- orendoglycosidase. The glycosidase is typically a mutant, which isengineered to form glycosyl bonds rather than cleave them. The mutantglycanase typically includes a substitution of an amino acid residue foran active site acidic amino acid residue. For example, when theendoglycanase is endo-H, the substituted active site residues willtypically be Asp at position 130, Glu at position 132 or a combinationthereof. The amino acids are generally replaced with serine, alanine,asparagine, or glutamine.

The mutant enzyme catalyzes the reaction, usually by a synthesis stepthat is analogous to the reverse reaction of the endoglycanasehydrolysis step. In these embodiments, the glycosyl donor molecule(e.g., a desired oligo- or mono-saccharide structure) contains a leavinggroup and the reaction proceeds with the addition of the donor moleculeto a GlcNAc residue on the protein. For example, the leaving group canbe a halogen, such as fluoride. In other embodiments, the leaving groupis a Asn, or a Asn-peptide moiety. In yet further embodiments, theGlcNAc residue on the glycosyl donor molecule is modified. For example,the GlcNAc residue may comprise a 1,2 oxazoline moiety.

In another embodiment, each of the enzymes utilized to produce aconjugate of the invention are present in a catalytic amount. Thecatalytic amount of a particular enzyme varies according to theconcentration of that enzyme's substrate as well as to reactionconditions such as temperature, time and pH value. Means for determiningthe catalytic amount for a given enzyme under preselected substrateconcentrations and reaction conditions are well known to those of skillin the art.

The temperature at which an above process is carried out can range fromjust above freezing to the temperature at which the most sensitiveenzyme denatures. Preferred temperature ranges are about 0° C. to about55° C., and more preferably about 20° C. to about 30° C. In anotherexemplary embodiment, one or more components of the present method areconducted at an elevated temperature using a thermophilic enzyme.

The reaction mixture is maintained for a period of time sufficient forthe acceptor to be glycosylated, thereby forming the desired conjugate.Some of the conjugate can often be detected after a few hours, withrecoverable amounts usually being obtained within 24 hours or less.Those of skill in the art understand that the rate of reaction isdependent on a number of variable factors (e.g, enzyme concentration,donor concentration, acceptor concentration, temperature, solventvolume), which are optimized for a selected system.

The present invention also provides for the industrial-scale productionof modified peptides. As used herein, an industrial scale generallyproduces at least about 250 mg, preferably at least about 500 mg, andmore preferably at least about 1 gram of finished, purified conjugate,preferably after a single reaction cycle, i.e., the conjugate is not acombination the reaction products from identical, consecutively iteratedsynthesis cycles.

In the discussion that follows, the invention is exemplified by theconjugation of modified sialic acid moieties to a glycosylated peptide.The exemplary modified sialic acid is labeled with (m-) PEG. The focusof the following discussion on the use of PEG-modified sialic acid andglycosylated peptides is for clarity of illustration and is not intendedto imply that the invention is limited to the conjugation of these twopartners. One of skill understands that the discussion is generallyapplicable to the additions of modified glycosyl moieties other thansialic acid. Moreover, the discussion is equally applicable to themodification of a glycosyl unit with agents other than PEG includingother water-soluble polymers, therapeutic moieties, and biomolecules.

An enzymatic approach can be used for the selective introduction of (m-)PEG-ylated or (m-) PPG-ylated carbohydrates onto a peptide orglycopeptide. The method utilizes modified sugars containing PEG, PPG,or a masked reactive functional group, and is combined with theappropriate glycosyltransferase or glycosynthase. By selecting theglycosyltransferase that will make the desired carbohydrate linkage andutilizing the modified sugar as the donor substrate, the PEG or PPG canbe introduced directly onto the peptide backbone, onto existing sugarresidues of a glycopeptide or onto sugar residues that have been addedto a peptide.

An acceptor for the sialyltransferase is present on the peptide to bemodified by the methods of the present invention either as a naturallyoccurring structure or one placed there recombinantly, enzymatically orchemically. Suitable acceptors, include, for example, galactosylacceptors such as GalNAc, Galβ-1,4GlcNAc, Galβ-1,4GalNAc, Galβ1,3GalNAc,lacto-N-tetraose, Galβ1,3GlcNAc, Galβ-1,3Ara, Galβ-1,6GlcNAc, Galβ1,4Glc(lactose), and other acceptors known to those of skill in the art (see,e.g., Paulson et al., J. Biol. Chem. 253: 5617-5624 (1978)).

In one embodiment, an acceptor for the sialyltransferase is present onthe glycopeptide to be modified upon in vivo synthesis of theglycopeptide. Such glycopeptides can be sialylated using the claimedmethods without prior modification of the glycosylation pattern of theglycopeptide. Alternatively, the methods of the invention can be used tosialylate a peptide that does not include a suitable acceptor; one firstmodifies the peptide to include an acceptor by methods known to those ofskill in the art. In an exemplary embodiment, a GalNAc residue is addedto an O-linked glycosylation site by the action of a GalNAc transferase.Hassan H, Bennett E P, Mandel U, Hollingsworth Mass., and Clausen H(2000). Control of Mucin-Type O-Glycosylation: O-Glycan Occupancy isDirected by Substrate Specificities of Polypeptide GalNAc-Transferases.(Eds. Ernst, Hart, and Sinay). Wiley-VCH chapter “Carbohydrates inChemistry and Biology—a Comprehension Handbook”, 273-292.

In an exemplary embodiment, the galactosyl acceptor is assembled byattaching a galactose residue to an appropriate acceptor linked to thepeptide, e.g., a GalNAc. The method includes incubating the peptide tobe modified with a reaction mixture that contains a suitable amount of agalactosyltransferase (e.g., Galβ1,3 or Galβ1,4), and a suitablegalactosyl donor (e.g., UDP-galactose). The reaction is allowed toproceed substantially to completion or, alternatively, the reaction isterminated when a preselected amount of the galactose residue is added.Other methods of assembling a selected saccharide acceptor will beapparent to those of skill in the art.

In yet another embodiment, glycopeptide-linked oligosaccharides arefirst “trimmed,” either in whole or in part, to expose either anacceptor for the sialyltransferase or a moiety to which one or moreappropriate residues can be added to obtain a suitable acceptor. Enzymessuch as glycosyltransferases and endoglycosidases (see, for example U.S.Pat. No. 5,716,812) are useful for the attaching and trimming reactions.

In the discussion that follows, the method of the invention isexemplified by the use of modified sugars having a water-soluble polymerattached thereto. The focus of the discussion is for clarity ofillustration. Those of skill will appreciate that the discussion isequally relevant to those embodiments in which the modified sugar bearsa therapeutic moiety, biomolecule or the like.

In an exemplary embodiment, an O-linked carbohydrate residue is“trimmed” prior to the addition of the modified sugar. For example aGalNAc-Gal residue is trimmed back to GalNAc. A modified sugar bearing awater-soluble polymer is conjugated to one or more of the sugar residuesexposed by the “trimming.” In one example, a glycopeptide is “trimmed”and a water-soluble polymer is added to the resulting O-side chain aminoacid or glycopeptide glycan via a saccharyl moiety, e.g., Sia, Gal orGalNAc moiety conjugated to the water-soluble polymer. The modifiedsaccharyl moiety is attached to an acceptor site on the “trimmed”glycopeptide. Alternatively, an unmodified saccharyl moiety, e.g., Galcan be added the terminus of the O-linked glycan.

In another exemplary embodiment, a water-soluble polymer is added to aGalNAc residue via a modified sugar having a galactose residue.Alternatively, an unmodified Gal can be added to the terminal GalNAcresidue.

In yet a further example, a water-soluble polymer is added onto a Galresidue using a modified sialic acid.

In another exemplary embodiment, an O-linked glycosyl residue is“trimmed back” to the GalNAc attached to the amino acid. In one example,a water-soluble polymer is added via a Gal modified with the polymer.Alternatively, an unmodified Gal is added to the GalNAc, followed by aGal with an attached water-soluble polymer. In yet another embodiment,one or more unmodified Gal residue is added to the GalNAc, followed by asialic acid moiety modified with a water-soluble polymer.

The exemplary embodiments discussed above provide an illustration of thepower of the methods set forth herein. Using the methods of theinvention, it is possible to “trim back” and build up a carbohydrateresidue of substantially any desired structure. The modified sugar canbe added to the termini of the carbohydrate moiety as set forth above,or it can be intermediate between the peptide core and the terminus ofthe carbohydrate.

In an exemplary embodiment, the water-soluble polymer is added to aterminal Gal residue using a polymer modified sialic acid. Anappropriate sialyltransferase is used to add a modified sialic acid. Theapproach is summarized in Scheme 5.

In yet a further approach, summarized in Scheme 6, a masked reactivefunctionality is present on the sialic acid. The masked reactive groupis preferably unaffected by the conditions used to attach the modifiedsialic acid to the peptide. After the covalent attachment of themodified sialic acid to the peptide, the mask is removed and the peptideis conjugated with an agent such as PEG, PPG, a therapeutic moiety,biomolecule or other agent. The agent is conjugated to the peptide in aspecific manner by its reaction with the unmasked reactive group on themodified sugar residue.

Any modified sugar can be used with its appropriate glycosyltransferase,depending on the terminal sugars of the oligosaccharide side chains ofthe glycopeptide (Table 3). As discussed above, the terminal sugar ofthe glycopeptide required for introduction of the PEG-ylated orPPGylated structure can be introduced naturally during expression or itcan be produced post expression using the appropriate glycosidase(s),glycosyltransferase(s) or mix of glycosidase(s) andglycosyltransferase(s).

TABLE 3

X = O, NH, S, CH₂, N—(R₁₋₅)₂. Y = X; Z = X; A = X; B = X. Q = H₂, O, S,NH, N—R. R, R₁₋₄ = H, Linker-M, M. M = Ligand of interest Ligand ofinterest = acyl-PEG, acyl-PPG, alkyl-PEG, acyl-alkyl-PEG,acyl-alkyl-PEG, carbamoyl-PEG, carbamoyl-PPG, PEG, PPG, acyl-aryl-PEG,acyl-aryl-PPG, aryl-PEG, aryl-PPG, Mannose-₆-phosphate, heparin,heparan, SLex, Mannose, FGF, VFGF, protein, chondroitin, keratan,dermatan, albumin, integrins, peptides, etc.

In an alternative embodiment, the modified sugar is added directly tothe peptide backbone using a glycosyltransferase known to transfer sugarresidues to the O-linked glycosylation site on the peptide backbone.This exemplary embodiment is set forth in Scheme 7. Exemplaryglycosyltransferases useful in practicing the present invention include,but are not limited to, GalNAc transferases (GalNAc T1-20), GlcNActransferases, fucosyltransferases, glucosyltransferases,xylosyltransferases, mannosyltransferases and the like. Use of thisapproach allows the direct addition of modified sugars onto peptidesthat lack any carbohydrates or, alternatively, onto existingglycopeptides. In both cases, the addition of the modified sugar occursat specific positions on the peptide backbone as defined by thesubstrate specificity of the glycosyltransferase and not in a randommanner as occurs during modification of a protein's peptide backboneusing chemical methods. An array of agents can be introduced intoproteins or glycopeptides that lack the glycosyltransferase substratepeptide sequence by engineering the appropriate amino acid sequence intothe polypeptide chain.

In each of the exemplary embodiments set forth above, one or moreadditional chemical or enzymatic modification steps can be utilizedfollowing the conjugation of the modified sugar to the peptide. In anexemplary embodiment, an enzyme (e.g., fucosyltransferase) is used toappend a glycosyl unit (e.g., fucose) onto the terminal modified sugarattached to the peptide. In another example, an enzymatic reaction isutilized to “cap” (e.g., sialylate) sites to which the modified sugarfailed to conjugate. Alternatively, a chemical reaction is utilized toalter the structure of the conjugated modified sugar. For example, theconjugated modified sugar is reacted with agents that stabilize ordestabilize its linkage with the peptide component to which the modifiedsugar is attached. In another example, a component of the modified sugaris deprotected following its conjugation to the peptide. One of skillwill appreciate that there is an array of enzymatic and chemicalprocedures that are useful in the methods of the invention at a stageafter the modified sugar is conjugated to the peptide. Furtherelaboration of the modified sugar-peptide conjugate is within the scopeof the invention.

In another exemplary embodiment, the glycopeptide is conjugated to atargeting agent, e.g., transferrin (to deliver the peptide across theblood-brain barrier, and to endosomes), carnitine (to deliver thepeptide to muscle cells; see, for example, LeBorgne et al., Biochem.Pharmacol. 59: 1357-63 (2000), and phosphonates, e.g., bisphosphonate(to target the peptide to bone and other calciferous tissues; see, forexample, Modern Drug Discovery, August 2002, page 10). Other agentsuseful for targeting are apparent to those of skill in the art. Forexample, glucose, glutamine and IGF are also useful to target muscle.

The targeting moiety and therapeutic peptide are conjugated by anymethod discussed herein or otherwise known in the art. Those of skillwill appreciate that peptides in addition to those set forth above canalso be derivatized as set forth herein. Exemplary peptides are setforth in the Appendix attached to copending, commonly owned U.S.Provisional Patent Application No. 60/328,523 filed Oct. 10, 2001.

In an exemplary embodiment, the targeting agent and the therapeuticpeptide are coupled via a linker moiety. In this embodiment, at leastone of the therapeutic peptide or the targeting agent is coupled to thelinker moiety via an intact glycosyl linking group according to a methodof the invention. In an exemplary embodiment, the linker moiety includesa poly(ether) such as poly(ethylene glycol). In another exemplaryembodiment, the linker moiety includes at least one bond that isdegraded in vivo, releasing the therapeutic peptide from the targetingagent, following delivery of the conjugate to the targeted tissue orregion of the body.

In yet another exemplary embodiment, the in vivo distribution of thetherapeutic moiety is altered via altering a glycoform on thetherapeutic moiety without conjugating the therapeutic peptide to atargeting moiety. For example, the therapeutic peptide can be shuntedaway from uptake by the reticuloendothelial system by capping a terminalgalactose moiety of a glycosyl group with sialic acid (or a derivativethereof).

i. Enzymes

1. Glycosyltransferases

Glycosyltransferases catalyze the addition of activated sugars (donorNDP-sugars), in a step-wise fashion, to a protein, glycopeptide, lipidor glycolipid or to the non-reducing end of a growing oligosaccharide.N-linked glycopeptides are synthesized via a transferase and alipid-linked oligosaccharide donor Dol-PP-NAG₂Glc₃Mang in an en blocktransfer followed by trimming of the core. In this case the nature ofthe “core” saccharide is somewhat different from subsequent attachments.A very large number of glycosyltransferases are known in the art.

The glycosyltransferase to be used in the present invention may be anyas long as it can utilize the modified sugar as a sugar donor. Examplesof such enzymes include Leloir pathway glycosyltransferase, such asgalactosyltransferase, N-acetylglucosaminyltransferase,N-acetylgalactosaminyltransferase, fucosyltransferase,sialyltransferase, mannosyltransferase, xylosyltransferase,glucurononyltransferase and the like.

For enzymatic saccharide syntheses that involve glycosyltransferasereactions, glycosyltransferase can be cloned, or isolated from anysource. Many cloned glycosyltransferases are known, as are theirpolynucleotide sequences. See, e.g., “The WWW Guide To ClonedGlycosyltransferases,” (http://www.vei.co.uk/TGN/gt guide.htm).Glycosyltransferase amino acid sequences and nucleotide sequencesencoding glycosyltransferases from which the amino acid sequences can bededuced are also found in various publicly available databases,including GenBank, Swiss-Prot, EMBL, and others.

Glycosyltransferases that can be employed in the methods of theinvention include, but are not limited to, galactosyltransferases,fucosyltransferases, glucosyltransferases,N-acetylgalactosaminyltransferases, N-acetylglucosaminyltransferases,glucuronyltransferases, sialyltransferases, mannosyltransferases,glucuronic acid transferases, galacturonic acid transferases, andoligosaccharyltransferases. Suitable glycosyltransferases include thoseobtained from eukaryotes, as well as from prokaryotes.

DNA encoding glycosyltransferases may be obtained by chemical synthesis,by screening reverse transcripts of mRNA from appropriate cells or cellline cultures, by screening genomic libraries from appropriate cells, orby combinations of these procedures. Screening of mRNA or genomic DNAmay be carried out with oligonucleotide probes generated from theglycosyltransferases gene sequence. Probes may be labeled with adetectable group such as a fluorescent group, a radioactive atom or achemiluminescent group in accordance with known procedures and used inconventional hybridization assays. In the alternative,glycosyltransferases gene sequences may be obtained by use of thepolymerase chain reaction (PCR) procedure, with the PCR oligonucleotideprimers being produced from the glycosyltransferases gene sequence. See,U.S. Pat. No. 4,683,195 to Mullis et al. and U.S. Pat. No. 4,683,202 toMullis.

The glycosyltransferase may be synthesized in host cells transformedwith vectors containing DNA encoding the glycosyltransferases enzyme.Vectors are used either to amplify DNA encoding the glycosyltransferasesenzyme and/or to express DNA which encodes the glycosyltransferasesenzyme. An expression vector is a replicable DNA construct in which aDNA sequence encoding the glycosyltransferases enzyme is operably linkedto suitable control sequences capable of effecting the expression of theglycosyltransferases enzyme in a suitable host. The need for suchcontrol sequences will vary depending upon the host selected and thetransformation method chosen. Generally, control sequences include atranscriptional promoter, an optional operator sequence to controltranscription, a sequence encoding suitable mRNA ribosomal bindingsites, and sequences which control the termination of transcription andtranslation. Amplification vectors do not require expression controldomains. All that is needed is the ability to replicate in a host,usually conferred by an origin of replication, and a selection gene tofacilitate recognition of transformants.

In an exemplary embodiment, the invention utilizes a prokaryotic enzyme.Such glycosyltransferases include enzymes involved in synthesis oflipooligosaccharides (LOS), which are produced by many gram negativebacteria (Preston et al., Critical Reviews in Microbiology 23(3):139-180 (1996)). Such enzymes include, but are not limited to, theproteins of the rfa operons of species such as E. coli and Salmonellatyphimurium, which include a β1,6 galactosyltransferase and a β1,3galactosyltransferase (see, e.g., EMBL Accession Nos. M80599 and M86935(E. coli); EMBL Accession No. S56361 (S. typhimurium)), aglucosyltransferase (Swiss-Prot Accession No. P25740 (E. coli), anβ1,2-glucosyltransferase (rfaJ)(Swiss-Prot Accession No. P27129 (E.coli) and Swiss-Prot Accession No. P19817 (S. typhimurium)), and an1,2-N-acetylglucosaminyltransferase (rfaK)(EMBL Accession No. U00039 (E.coli). Other glycosyltransferases for which amino acid sequences areknown include those that are encoded by operons such as rfaB, which havebeen characterized in organisms such as Klebsiella pneumoniae, E. coli,Salmonella typhimurium, Salmonella enterica, Yersinia enterocolitica,Mycobacterium leprosum, and the rh1 operon of Pseudomonas aeruginosa.

Also suitable for use in the present invention are glycosyltransferasesthat are involved in producing structures containinglacto-N-neotetraose,D-galactosyl-β-1,4-N-acetyl-D-glucosaminyl-β-1,3-D-galactosyl-β-1,4-D-glucose,and the P^(k) blood group trisaccharide sequence,D-galactosyl-α-1,4-D-galactosyl-β-1,4-D-glucose, which have beenidentified in the LOS of the mucosal pathogens Neisseria gonnorhoeae andN. meningitidis (Scholten et al., J. Med. Microbiol. 41: 236-243(1994)). The genes from N. meningitidis and N. gonorrhoeae that encodethe glycosyltransferases involved in the biosynthesis of thesestructures have been identified from N. meningitidis immunotypes L3 andLi (Jennings et al., Mol. Microbiol. 18: 729-740 (1995)) and the N.gonorrhoeae mutant F62 (Gotshlich, J. Exp. Med. 180: 2181-2190 (1994)).In N. meningitidis, a locus consisting of three genes, lgtA, lgtB and lgE, encodes the glycosyltransferase enzymes required for addition of thelast three of the sugars in the lacto-N-neotetraose chain (Wakarchuk etal., J. Biol. Chem. 271: 19166-73 (1996)). Recently the enzymaticactivity of the lgtB and lgtA gene product was demonstrated, providingthe first direct evidence for their proposed glycosyltransferasefunction (Wakarchuk et al., J. Biol. Chem. 271(45): 28271-276 (1996)).In N. gonorrhoeae, there are two additional genes, IgtD which addsβ-D-GalNAc to the 3 position of the terminal galactose of thelacto-N-neotetraose structure and lgtC which adds a terminal α-D-Gal tothe lactose element of a truncated LOS, thus creating the pk blood groupantigen structure (Gotshlich (1994), supra.). In N. meningitidis, aseparate immunotype L1 also expresses the P^(k) blood group antigen andhas been shown to carry an IgtC gene (Jennings et al., (1995), supra.).Neisseria glycosyltransferases and associated genes are also describedin U.S. Pat. No. 5,545,553 (Gotschlich). Genes forα1,2-fucosyltransferase and α1,3-fucosyltransferase from Helicobacterpylori has also been characterized (Martin et al., J. Biol. Chem. 272:21349-21356 (1997)). Also of use in the present invention are theglycosyltransferases of Campylobacter jejuni (see, for example,http://afmb.cnrs-mrs.fr/pedro/CAZY/gtf_(—)42.html).

a) Fucosyltransferases

In some embodiments, a glycosyltransferase used in the method of theinvention is a fucosyltransferase. Fucosyltransferases are known tothose of skill in the art. Exemplary fucosyltransferases includeenzymes, which transfer L-fucose from GDP-fucose to a hydroxy positionof an acceptor sugar. Fucosyltransferases that transfer non-nucleotidesugars to an acceptor are also of use in the present invention.

In some embodiments, the acceptor sugar is, for example, the GlcNAc in aGalβ(1→3,4)GlcNAcβ-group in an oligosaccharide glycoside. Suitablefucosyltransferases for this reaction include theGalβ(1→3,4)GlcNAcβ1-α(1→3,4)fucosyltransferase (FTIII E.C. No.2.4.1.65), which was first characterized from human milk (see, Palcic,et al., Carbohydrate Res. 190:1-11 (1989); Prieels, et al., J. Biol.Chem. 256: 10456-10463 (1981); and Nunez, et al., Can. J. Chem. 59:2086-2095 (1981)) and the Galβ(1→3,4)GlcNAcβ-αfucosyltransferases (FTIV,FTV, FTVI) which are found in human serum. FTVII (E.C. No. 2.4.1.65), asialyl α(2→3)Galβ((1→3)GlcNAcβ-fucosyltransferase, has also beencharacterized. A recombinant form of the Galβ(1→3,4)GlcNAcβ-α(1→3,4)fucosyltransferase has also been characterized (see,Dumas, et al., Bioorg. Med. Letters 1: 425-428 (1991) andKukowska-Latallo, et al., Genes and Development 4: 1288-1303 (1990)).Other exemplary fucosyltransferases include, for example, α1,2fucosyltransferase (E.C. No. 2.4.1.69). Enzymatic fucosylation can becarried out by the methods described in Mollicone, et al., Eur. J.Biochem. 191: 169-176 (1990) or U.S. Pat. No. 5,374,655. Cells that areused to produce a fucosyltransferase will also include an enzymaticsystem for synthesizing GDP-fucose.

b) Galactosyltransferases

In another group of embodiments, the glycosyltransferase is agalactosyltransferase. Exemplary galactosyltransferases include α(1,3)galactosyltransferases (E.C. No. 2.4.1.151, see, e.g., Dabkowski et al.,Transplant Proc. 25:2921 (1993) and Yamamoto et al. Nature 345: 229-233(1990), bovine (GenBank jO4989, Joziasse et al., J. Biol. Chem. 264:14290-14297 (1989)), murine (GenBank m26925; Larsen et al., Proc. Nat'l.Acad. Sci. USA 86: 8227-8231 (1989)), porcine (GenBank L36152; Strahanet al., Immunogenetics 41: 101-105 (1995)). Another suitable α1,3galactosyltransferase is that which is involved in synthesis of theblood group B antigen (EC 2.4.1.37, Yamamoto et al., J. Biol. Chem. 265:1146-1151 (1990) (human)). Yet a further exemplary galactosyltransferaseis core Gal-T1.

Also suitable for use in the methods of the invention are β(1,4)galactosyltransferases, which include, for example, EC 2.4.1.90 (LacNAcsynthetase) and EC 2.4.1.22 (lactose synthetase) (bovine (D'Agostaro etal., Eur. J. Biochem. 183: 211-217 (1989)), human (Masri et al.,Biochem. Biophys. Res. Commun. 157: 657-663 (1988)), murine (Nakazawa etal., J. Biochem. 104: 165-168 (1988)), as well as E.C. 2.4.1.38 and theceramide galactosyltransferase (EC 2.4.1.45, Stahl et al., J. Neurosci.Res. 38: 234-242 (1994)). Other suitable galactosyltransferases include,for example, α1,2 galactosyltransferases (from e.g., Schizosaccharomycespombe, Chapell et al., Mol. Biol. Cell 5: 519-528 (1994)).

Also suitable in the practice of the invention are r soluble forms ofα1,3-galactosyltransferase such as that reported by Cho, S. K. andCummings, R. D. (1997) J. Biol. Chem., 272, 13622-13628.

c) Sialyltransferases

Sialyltransferases are another type of glycosyltransferase that isuseful in the recombinant cells and reaction mixtures of the invention.Cells that produce recombinant sialyltransferases will also produceCMP-sialic acid, which is a sialic acid donor for sialyltransferases.Examples of sialyltransferases that are suitable for use in the presentinvention include ST3Gal III (e.g., a rat or human ST3Gal III), ST3GalIV, ST3Gal I, ST6Gal I, ST3Gal V, ST6Gal II, ST6GalNAc I, ST6GalNAc II,and ST6GalNAc III (the sialyltransferase nomenclature used herein is asdescribed in Tsuji et al., Glycobiology 6: v-xiv (1996)). An exemplaryα(2,3)sialyltransferase referred to as α(2,3)sialyltransferase (EC2.4.99.6) transfers sialic acid to the non-reducing terminal Gal of aGalβ1→3Glc disaccharide or glycoside. See, Van den Eijnden et al., J.Biol. Chem. 256: 3159 (1981), Weinstein et al., J. Biol. Chem. 257:13845 (1982) and Wen et al., J. Biol. Chem. 267: 21011 (1992). Anotherexemplary α-2,3-sialyltransferase (EC 2.4.99.4) transfers sialic acid tothe non-reducing terminal Gal of the disaccharide or glycoside. see,Rearick et al., J. Biol. Chem. 254: 4444 (1979) and Gillespie et al., J.Biol. Chem. 267: 21004 (1992). Further exemplary enzymes includeGal-β-1,4-GlcNAc α-2,6 sialyltransferase (See, Kurosawa et al. Eur. J.Biochem. 219: 375-381 (1994)).

Preferably, for glycosylation of carbohydrates of glycopeptides thesialyltransferase will be able to transfer sialic acid to the sequenceGalβ1,4GlcNAc-, the most common penultimate sequence underlying theterminal sialic acid on fully sialylated carbohydrate structures (see,Table 5).

TABLE 5 Sialyltransferases which use the Galβ1,4GlcNAc sequence as anacceptor substrate Sialyltransferase Source Sequence(s) formed Ref.ST6Gal I Mammalian NeuAcα2,6Galβ1,4GlcNAc- 1 ST3Gal III MammalianNeuAcα2,3Galβ1,4GlcNAc- 1 NeuAcα2,3Galβ1,3GlcNAc- ST3Gal IV MammalianNeuAcα2,3Galβ1,4GlcNAc- 1 NeuAcα2,3Galβ1,3GlcNAc- ST6Gal II MammalianNeuAcα2,6Galβ1,4GlcNAc ST6Gal II photobacterium NeuAcα2,6Galβ1,4GlcNAc-2 ST3Gal V N. meningitides NeuAcα2,3Galβ1,4GlcNAc- 3 N. gonorrhoeae 1)Goochee et al, Bio/Technology 9: 1347-1355 (1991) 2) Yamamoto et al, J.Biochem. 120: 104-110 (1996) 3) Gilbert et al., J. Biol. Chem. 271:28271-28276 (1996)

An example of a sialyltransferase that is useful in the claimed methodsis ST3Gal III, which is also referred to as α(2,3)sialyltransferase (EC2.4.99.6). This enzyme catalyzes the transfer of sialic acid to the Galof a Galβ1,3GlcNAc or Galβ1,4GlcNAc glycoside (see, e.g., Wen et al., J.Biol. Chem. 267: 21011 (1992); Van den Eijnden et al., J. Biol. Chem.256: 3159 (1991)) and is responsible for sialylation ofasparagine-linked oligosaccharides in glycopeptides. The sialic acid islinked to a Gal with the formation of an α-linkage between the twosaccharides. Bonding (linkage) between the saccharides is between the2-position of NeuAc and the 3-position of Gal. This particular enzymecan be isolated from rat liver (Weinstein et al., J. Biol. Chem. 257:13845 (1982)); the human cDNA (Sasaki et al. (1993) J. Biol. Chem. 268:22782-22787; Kitagawa & Paulson (1994) J. Biol. Chem. 269:1394-1401) andgenomic (Kitagawa et al. (1996) J. Biol. Chem. 271: 931-938) DNAsequences are known, facilitating production of this enzyme byrecombinant expression. In another embodiment, the claimed sialylationmethods use a rat ST3Gal III.

Other exemplary sialyltransferases of use in the present inventioninclude those isolated from Campylobacter jejuni, including the α(2,3).See, e.g, WO99/49051.

Sialyltransferases other those listed in Table 5, are also useful in aneconomic and efficient large-scale process for sialylation ofcommercially important glycopeptides. As a simple test to find out theutility of these other enzymes, various amounts of each enzyme (1-100mU/mg protein) are reacted with asialo-α₁ AGP (at 1-10 mg/ml) to comparethe ability of the sialyltransferase of interest to sialylateglycopeptides relative to either bovine ST6Gal I, ST3Gal III or bothsialyltransferases. Alternatively, other glycopeptides or glycopeptides,or N-linked oligosaccharides enzymatically released from the peptidebackbone can be used in place of asialo-α₁ AGP for this evaluation.Sialyltransferases with the ability to sialylate N-linkedoligosaccharides of glycopeptides more efficiently than ST6Gal I areuseful in a practical large-scale process for peptide sialylation (asillustrated for ST3Gal III in this disclosure). Other exemplarysialyltransferases are shown in FIG. 10.

d) GalNAc Transferases

N-acetylgalactosaminyltransferases are of use in practicing the presentinvention, particularly for binding a GalNAc moiety to an amino acid ofthe O-linked glycosylation site of the peptide. SuitableN-acetylgalactosaminyltransferases include, but are not limited to,α(1,3) N-acetylgalactosaminyltransferase, β(1,4)N-acetylgalactosaminyltransferases (Nagata et al., J. Biol. Chem. 267:12082-12089 (1992) and Smith et al., J. Biol. Chem. 269: 15162 (1994))and polypeptide N-acetylgalactosaminyltransferase (Homa et al., J. Biol.Chem. 268: 12609 (1993)).

Production of proteins such as the enzyme GalNAc T_(I-XX) from clonedgenes by genetic engineering is well known. See, e.g., U.S. Pat. No.4,761,371. One method involves collection of sufficient samples, thenthe amino acid sequence of the enzyme is determined by N-terminalsequencing. This information is then used to isolate a cDNA cloneencoding a full-length (membrane bound) transferase which uponexpression in the insect cell line Sf9 resulted in the synthesis of afully active enzyme. The acceptor specificity of the enzyme is thendetermined using a semiquantitative analysis of the amino acidssurrounding known glycosylation sites in 16 different proteins followedby in vitro glycosylation studies of synthetic peptides. This work hasdemonstrated that certain amino acid residues are over represented inglycosylated peptide segments and that residues in specific positionssurrounding glycosylated serine and threonine residues may have a moremarked influence on acceptor efficiency than other amino acid moieties.

2. Sulfotransferases

The invention also provides methods for producing peptides that includesulfated molecules, including, for example sulfated polysaccharides suchas heparin, heparan sulfate, carragenen, and related compounds. Suitablesulfotransferases include, for example, chondroitin-6-sulphotransferase(chicken cDNA described by Fukuta et al., J. Biol. Chem. 270:18575-18580 (1995); GenBank Accession No. D49915), glycosaminoglycanN-acetylglucosamine N-deacetylase/N-sulphotransferase 1 (Dixon et al.,Genomics 26: 239-241 (1995); UL 18918), and glycosaminoglycanN-acetylglucosamine N-deacetylase/N-sulphotransferase 2 (murine cDNAdescribed in Orellana et al., J. Biol. Chem. 269: 2270-2276 (1994) andEriksson et al., J. Biol. Chem. 269: 10438-10443 (1994); human cDNAdescribed in GenBank Accession No. U2304).

3. Cell-Bound Glycosyltransferases

In another embodiment, the enzymes utilized in the method of theinvention are cell-bound glycosyltransferases. Although many solubleglycosyltransferases are known (see, for example, U.S. Pat. No.5,032,519), glycosyltransferases are generally in membrane-bound formwhen associated with cells. Many of the membrane-bound enzymes studiedthus far are considered to be intrinsic proteins; that is, they are notreleased from the membranes by sonication and require detergents forsolubilization. Surface glycosyltransferases have been identified on thesurfaces of vertebrate and invertebrate cells, and it has also beenrecognized that these surface transferases maintain catalytic activityunder physiological conditions. However, the more recognized function ofcell surface glycosyltransferases is for intercellular recognition(Roth, MOLECULAR APPROACHES to SUPRACELLULAR PHENOMENA, 1990).

Methods have been developed to alter the glycosyltransferases expressedby cells. For example, Larsen et al., Proc. Natl. Acad. Sci. USA 86:8227-8231 (1989), report a genetic approach to isolate cloned cDNAsequences that determine expression of cell surface oligosaccharidestructures and their cognate glycosyltransferases. A cDNA librarygenerated from mRNA isolated from a murine cell line known to expressUDP-galactose:.β.-D-galactosyl-1,4-N-acetyl-D-glucosaminideα-1,3-galactosyltransferase was transfected into COS-1 cells. Thetransfected cells were then cultured and assayed for α 1-3galactosyltransferase activity.

Francisco et al., Proc. Natl. Acad. Sci. USA 89: 2713-2717 (1992),disclose a method of anchoring β-lactamase to the external surface ofEscherichia coli. A tripartite fusion consisting of (i) a signalsequence of an outer membrane protein, (ii) a membrane-spanning sectionof an outer membrane protein, and (iii) a complete mature β-lactamasesequence is produced resulting in an active surface bound β-lactamasemolecule. However, the Francisco method is limited only to procaryoticcell systems and as recognized by the authors, requires the completetripartite fusion for proper functioning.

4. Fusion Proteins

In other exemplary embodiments, the methods of the invention utilizefusion proteins that have more than one enzymatic activity that isinvolved in synthesis of a desired glycopeptide conjugate. The fusionpolypeptides can be composed of, for example, a catalytically activedomain of a glycosyltransferase that is joined to a catalytically activedomain of an accessory enzyme. The accessory enzyme catalytic domaincan, for example, catalyze a step in the formation of a nucleotide sugarthat is a donor for the glycosyltransferase, or catalyze a reactioninvolved in a glycosyltransferase cycle. For example, a polynucleotidethat encodes a glycosyltransferase can be joined, in-frame, to apolynucleotide that encodes an enzyme involved in nucleotide sugarsynthesis. The resulting fusion protein can then catalyze not only thesynthesis of the nucleotide sugar, but also the transfer of the sugarmoiety to the acceptor molecule. The fusion protein can be two or morecycle enzymes linked into one expressible nucleotide sequence. In otherembodiments the fusion protein includes the catalytically active domainsof two or more glycosyltransferases. See, for example, 5,641,668. Themodified glycopeptides of the present invention can be readily designedand manufactured utilizing various suitable fusion proteins (see, forexample, PCT Patent Application PCT/CA98/01180, which was published asWO 99/31224 on Jun. 24, 1999.)

5. Immobilized Enzymes

In addition to cell-bound enzymes, the present invention also providesfor the use of enzymes that are immobilized on a solid and/or solublesupport. In an exemplary embodiment, there is provided aglycosyltransferase that is conjugated to a PEG via an intact glycosyllinker according to the methods of the invention. The PEG-linker-enzymeconjugate is optionally attached to solid support. The use of solidsupported enzymes in the methods of the invention simplifies the work upof the reaction mixture and purification of the reaction product, andalso enables the facile recovery of the enzyme. The glycosyltransferaseconjugate is utilized in the methods of the invention. Othercombinations of enzymes and supports will be apparent to those of skillin the art.

Purification of Peptide Conjugates

The products produced by the above processes can be used withoutpurification. However, it is usually preferred to recover the product.Standard, well-known techniques for recovery of glycosylated saccharidessuch as thin or thick layer chromatography, column chromatography, ionexchange chromatography, or membrane filtration can be used. It ispreferred to use membrane filtration, more preferably utilizing areverse osmotic membrane, or one or more column chromatographictechniques for the recovery as is discussed hereinafter and in theliterature cited herein. For instance, membrane filtration wherein themembranes have molecular weight cutoff of about 3000 to about 10,000 canbe used to remove proteins such as glycosyl transferases. Nanofiltrationor reverse osmosis can then be used to remove salts and/or purify theproduct saccharides (see, e.g., WO 98/15581). Nanofilter membranes are aclass of reverse osmosis membranes that pass monovalent salts but retainpolyvalent salts and uncharged solutes larger than about 100 to about2,000 Daltons, depending upon the membrane used. Thus, in a typicalapplication, saccharides prepared by the methods of the presentinvention will be retained in the membrane and contaminating salts willpass through.

If the modified glycoprotein is produced intracellularly, as a firststep, the particulate debris, either host cells or lysed fragments, isremoved, for example, by centrifugation or ultrafiltration; optionally,the protein may be concentrated with a commercially available proteinconcentration filter, followed by separating the polypeptide variantfrom other impurities by one or more steps selected from immunoaffinitychromatography, ion-exchange column fractionation (e.g., ondiethylaminoethyl (DEAE) or matrices containing carboxymethyl orsulfopropyl groups), chromatography on Blue-Sepharose, CMBlue-Sepharose, MONO-Q, MONO—S, lentil lectin-Sepharose, WGA-Sepharose,Con A-Sepharose, Ether Toyopearl, Butyl Toyopearl, Phenyl Toyopearl,SP-Sepharose, or protein A Sepharose, SDS-PAGE chromatography, silicachromatography, chromatofocusing, reverse phase HPLC (e.g., silica gelwith appended aliphatic groups), gel filtration using, e.g., Sephadexmolecular sieve or size-exclusion chromatography, chromatography oncolumns that selectively bind the polypeptide, and ethanol or ammoniumsulfate precipitation.

Modified glycopeptides produced in culture are usually isolated byinitial extraction from cells, enzymes, etc., followed by one or moreconcentration, salting-out, aqueous ion-exchange, or size-exclusionchromatography steps, e.g., SP Sepharose. Additionally, the modifiedglycoprotein may be purified by affinity chromatography. HPLC may alsobe employed for one or more purification steps.

A protease inhibitor, e.g., methylsulfonylfluoride (PMSF) may beincluded in any of the foregoing steps to inhibit proteolysis andantibiotics may be included to prevent the growth of adventitiouscontaminants.

Within another embodiment, supernatants from systems which sproduce themodified glycopeptide of the invention are first concentrated using acommercially available protein concentration filter, for example, anAmicon or Millipore Pellicon ultrafiltration unit. Following theconcentration step, the concentrate may be applied to a suitablepurification matrix. For example, a suitable affinity matrix maycomprise a ligand for the peptide, a lectin or antibody molecule boundto a suitable support. Alternatively, an anion-exchange resin may beemployed, for example, a matrix or substrate having pendant DEAE groups.Suitable matrices include acrylamide, agarose, dextran, cellulose, orother types commonly employed in protein purification. Alternatively, acation-exchange step may be employed. Suitable cation exchangers includevarious insoluble matrices comprising sulfopropyl or carboxymethylgroups. Sulfopropyl groups are particularly preferred.

Finally, one or more RP-HPLC steps employing hydrophobic RP-HPLC media,e.g., silica gel having pendant methyl or other aliphatic groups, may beemployed to further purify a polypeptide variant composition. Some orall of the foregoing purification steps, in various combinations, canalso be employed to provide a homogeneous modified glycoprotein.

The modified glycopeptide of the invention resulting from a large-scalefermentation may be purified by methods analogous to those disclosed byUrdal et al., J. Chromatog. 296: 171 (1984). This reference describestwo sequential, RP-HPLC steps for purification of recombinant human IL-2on a preparative HPLC column. Alternatively, techniques such as affinitychromatography may be utilized to purify the modified glycoprotein.

Pharmaceutical Compositions

Polypeptides modified at various O-linked glycosylation site accordingto the method of the present invention have a broad range ofpharmaceutical applications. For example, modified erythropoietin (EPO)may be used for treating general anemia, aplastic anemia, chemo-inducedinjury (such as injury to bone marrow), chronic renal failure,nephritis, and thalassemia. Modified EPO may be further used fortreating neurological disorders such as brain/spine injury, multiplesclerosis, and Alzheimer's disease.

A second example is interferon-α (IFN-α), which may be used for treatingAIDS and hepatitis B or C, viral infections caused by a variety ofviruses such as human papilloma virus (HBV), coronavirus, humanimmunodeficiency virus (HIV), herpes simplex virus (HSV), andvaricella-zoster virus (VZV), cancers such as hairy cell leukemia,AIDS-related Kaposi's sarcoma, malignant melanoma, follicularnon-Hodgkins lymphoma, Philladephia chromosome (Ph)-positive, chronicphase myelogenous leukemia (CML), renal cancer, myeloma, chronicmyelogenous leukemia, cancers of the head and neck, bone cancers, aswell as cervical dysplasia and disorders of the central nervous system(CNS) such as multiple sclerosis. In addition, IFN-α modified accordingto the methods of the present invention is useful for treating anassortment of other diseases and conditions such as Sjogren's symdrome(an autoimmune disease), Behcet's disease (an autoimmune inflammatorydisease), fibromyalgia (a musculoskeletal pain/fatigue disorder),aphthous ulcer (canker sores), chronic fatigue syndrome, and pulmonaryfibrosis.

Another example is interferon-β, which is useful for treating CNSdisorders such as multiple sclerosis (either relapsing/remitting orchronic progressive), AIDS and hepatitis B or C, viral infections causedby a variety of viruses such as human papilloma virus (HBV), humanimmunodeficiency virus (HIV), herpes simplex virus (HSV), andvaricella-zoster virus (VZV), otological infections, musculoskeletalinfections, as well as cancers including breast cancer, brain cancer,colorectal cancer, non-small cell lung cancer, head and neck cancer,basal cell cancer, cervical dysplasia, melanoma, skin cancer, and livercancer. IFN-β modified according to the methods of the present inventionis also used in treating other diseases and conditions such astransplant rejection (e.g., bone marrow transplant), Huntington'schorea, colitis, brain inflammation, pulmonary fibrosis, maculardegeneration, hepatic cirrhosis, and keratoconjunctivitis.

Granulocyte colony stimulating factor (G-CSF) is a further example.G-CSF modified according to the methods of the present invention may beused as an adjunct in chemotherapy for treating cancers, and to preventor alleviate conditions or complications associated with certain medicalprocedures, e.g., chemo-induced bone marrow injury; leucopenia(general); chemo-induced febrile neutropenia; neutropenia associatedwith bone marrow transplants; and severe, chronic neutropenia. ModifiedG-CSF may also be used for transplantation; peripheral blood cellmobilization; mobilization of peripheral blood progenitor cells forcollection in patients who will receive myeloablative ormyelosuppressive chemotherapy; and reduction in duration of neutropenia,fever, antibiotic use, hospitalization following induction/consolidationtreatment for acute myeloid leukemia (AML). Other condictions ordisorders may be treated with modified G-CSF include asthma and allergicrhinitis.

As one additional example, human growth hormone (hGH) modified accordingto the methods of the present invention may be used to treatgrowth-related conditions such as dwarfism, short-stature in childrenand adults, cachexia/muscle wasting, general muscular atrophy, and sexchromosome abnormality (e.g., Turner's Syndrome). Other conditions maybe treated using modified hGH include: short-bowel syndrome,lipodystrophy, osteoporosis, uraemaia, burns, female infertility, boneregeneration, general diabetes, type II diabetes, osteo-arthritis,chronic obstructive pulmonary disease (COPD), and insomia. Moreover,modified hGH may also be used to promote various processes, e.g.,general tissue regeneration, bone regeneration, and wound healing, or asa vaccine adjunct.

Thus, in another aspect, the invention provides a pharmaceuticalcomposition. The pharmaceutical composition includes a pharmaceuticallyacceptable diluent and a covalent conjugate between anon-naturally-occurring, water-soluble polymer, therapeutic moiety orbiomolecule and a glycosylated or non-glycosylated peptide. The polymer,therapeutic moiety or biomolecule is conjugated to the peptide via anintact glycosyl linking group interposed between and covalently linkedto both the peptide and the polymer, therapeutic moiety or biomolecule.

Pharmaceutical compositions of the invention are suitable for use in avariety of drug delivery systems. Suitable formulations for use in thepresent invention are found in Remington's Pharmaceutical Sciences, MacePublishing Company, Philadelphia, Pa., 17th ed. (1985). For a briefreview of methods for drug delivery, see, Langer, Science 249:1527-1533(1990).

The pharmaceutical compositions may be formulated for any appropriatemanner of administration, including for example, topical, oral, nasal,intravenous, intracranial, intraperitoneal, subcutaneous orintramuscular administration. For parenteral administration, such assubcutaneous injection, the carrier preferably comprises water, saline,alcohol, a fat, a wax or a buffer. For oral administration, any of theabove carriers or a solid carrier, such as mannitol, lactose, starch,magnesium stearate, sodium saccharine, talcum, cellulose, glucose,sucrose, and magnesium carbonate, may be employed. Biodegradablematrises, such as microspheres (e.g., polylactate polyglycolate), mayalso be employed as carriers for the pharmaceutical compositions of thisinvention. Suitable biodegradable microspheres are disclosed, forexample, in U.S. Pat. Nos. 4,897,268 and 5,075,109.

Commonly, the pharmaceutical compositions are administeredsubcutaneously or parenterally, e.g., intravenously. Thus, the inventionprovides compositions for parenteral administration which comprise thecompound dissolved or suspended in an acceptable carrier, preferably anaqueous carrier, e.g., water, buffered water, saline, PBS and the like.The compositions may also contain detergents such as Tween 20 and Tween80; stabilizers such as mannitol, sorbitol, sucrose, and trehalose; andpreservatives such as EDTA and m-cresol. The compositions may containpharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents, tonicity adjusting agents, wetting agents, detergents and thelike.

These compositions may be sterilized by conventional sterilizationtechniques, or may be sterile filtered. The resulting aqueous solutionsmay be packaged for use as is, or lyophilized, the lyophilizedpreparation being combined with a sterile aqueous carrier prior toadministration. The pH of the preparations typically will be between 3and 11, more preferably from 5 to 9 and most preferably from 7 and 8.

In some embodiments the glycopeptides of the invention can beincorporated into liposomes formed from standard vesicle-forming lipids.A variety of methods are available for preparing liposomes, as describedin, e.g., Szoka et al., Ann. Rev. Biophys. Bioeng. 9: 467 (1980), U.S.Pat. Nos. 4,235,871, 4,501,728 and 4,837,028. The targeting of liposomesusing a variety of targeting agents (e.g., the sialyl galactosides ofthe invention) is well known in the art (see, e.g., U.S. Pat. Nos.4,957,773 and 4,603,044).

Standard methods for coupling targeting agents to liposomes can be used.These methods generally involve incorporation into liposomes of lipidcomponents, such as phosphatidylethanolamine, which can be activated forattachment of targeting agents, or derivatized lipophilic compounds,such as lipid-derivatized glycopeptides of the invention.

Targeting mechanisms generally require that the targeting agents bepositioned on the surface of the liposome in such a manner that thetarget moieties are available for interaction with the target, forexample, a cell surface receptor. The carbohydrates of the invention maybe attached to a lipid molecule before the liposome is formed usingmethods known to those of skill in the art (e.g., alkylation oracylation of a hydroxyl group present on the carbohydrate with a longchain alkyl halide or with a fatty acid, respectively). Alternatively,the liposome may be fashioned in such a way that a connector portion isfirst incorporated into the membrane at the time of forming themembrane. The connector portion must have a lipophilic portion, which isfirmly embedded and anchored in the membrane. It must also have areactive portion, which is chemically available on the aqueous surfaceof the liposome. The reactive portion is selected so that it will bechemically suitable to form a stable chemical bond with the targetingagent or carbohydrate, which is added later. In some cases it ispossible to attach the target agent to the connector molecule directly,but in most instances it is more suitable to use a third molecule to actas a chemical bridge, thus linking the connector molecule which is inthe membrane with the target agent or carbohydrate which is extended,three dimensionally, off of the vesicle surface.

The compounds prepared by the methods of the invention may also find useas diagnostic reagents. For example, labeled compounds can be used tolocate areas of inflammation or tumor metastasis in a patient suspectedof having an inflammation. For this use, the compounds can be labeledwith ¹²⁵I, ¹⁴C, or tritium.

The following examples are provided to illustrate the conjugates, andmethods and of the present invention, but not to limit the claimedinvention.

EXAMPLES Example 1 1.1a Preparation of Interferon alpha-2β-GalNAc (pH6.2)

Interferon alpha-2β was reconstituted by adding 200 μL water to 4 mg ofIFN alpha-2β. When the solid was dissolved, 1.92 mL reaction buffer (20mM MES, pH 6.2, 150 mM NaCl, 5 mM MgCl₂, 5 mM MnCl₂, 0.05% polysorbate,and 0.05% NaN₃), was added. UDP-GalNAc (4.16 mg; 3 mM) and GalNAc T2 (80mU; 80 μL) were then added and the reaction mixture was incubated at 32°C. with slow rotary movement. The reaction was monitored using MALDIanalysis and was essentially complete after 72 h

Once complete, the reaction mixture was submitted for peptide mapping,and analysis of site occupancy.

1.1b Preparation of Interferon Alpha-2β-GalNAc (pH 7.4).

The interferon alpha 2β was reconstituted as described by themanufacturer. Water, 50 μL, was added to 50 μg of IFN alpha-2β. When thesolid was dissolved, the reaction buffer (20 mM MES, pH 7.4, 150 mMNaCl, 5 mM MgCl₂, 5 mM MnCl₂, 0.05% polysorbate, and 0.05% NaN₃.), 50 Lwas added. The UDP-GalNAc (100 μg; 3 mM) and GalNAc T2 (8 mU; 8 μL) werethen added and the reaction mixture incubated at 32° C. under a slowrotary movement. The reaction was monitored using MALDI analysis and wasfound to be complete within about 48 to 72 h

1.2 Preparation of Interferon-Alpha-2β-GalNAc-SA-PEG-20 Kilodalton usingCMP-SA-PEG and ST6GalNAcI

The IFN-alpha-2β-GalNAc (1.0 mL, ˜2 mg, 0.1 mole) from 1.1 (above) wasbuffer exchanged (2×) using a 5 kilodalton MWCO Filter Centriconcartridge and a second buffer (20 mM MES, pH 7.4, 150 mM NaCl, 5 mMMgCl₂, 5 mM MnCl₂, 0.05% polysorbate, and 0.05% NaN₃). TheIFN-alpha-2β-GalNAc was reconstituted from the spin cartridge using thesecond buffer, 1.0 mL, and both CMP-SA-PEG-20 kilodalton (10 mg, 0.5micromoles) and ST6GalNAc 1 (200 μL) were added to the reaction mixture.The reaction was incubated at 32° C. for 96 h with slow rotary movement.The product, IFN-alpha-2β-GalNAc-SA-PEG-20 kilodalton was purified usingSP Sepharose and SEC (Superdex 75) chromatography. The addition ofsialic acid-PEG was verified using MALDI analysis.

1.3. Preparation of Interferon-Alpha-2β-GalNAc-Gal-SA-PEG-20 Kilodaltonusing CMP-SA-PEG, Core-1-β1,3-Galactosyl-Transferase, and ST3Gal2

The IFN-alpha-2β-GalNAc (1.0 mL, 2 mg, 0.1 mole) from the addition ofGalNAc described above (pH 6.2) was buffer exchanged (2×) using a 5kilodalton MWCO Filter Centricon cartridge and a second buffer (20 mMMES, pH 7.4, 150 mM NaCl, 5 mM MgCl₂, 5 mM MnCl₂, 0.05% polysorbate, and0.05% NaN₃). The IFN-alpha-2β-GalNAc was reconstituted from the spincartridge using 1.0 mL of the second buffer, containing CMP-SA-PEG-20kilodalton (10 mg, 0.5 micromoles), UDP-Galactose (1.8 mg, 3 mM),core-1-β1,3-galactosyl-transferase (200 mU on resin) and ST3Gal2 (200mU, α-2,3-(O)-sialytransferase). The reaction mixture was incubated at32° C. for 96 h with slow rotary movement. The product,IFN-alpha-2β-GalNAc-Gal-SA-PEG-20 kilodalton, was purified by SPSepharose and SEC (Superdex 75) chromatography. The addition of sialicacid-PEG was verified using MALDI analysis.

1.9 Protein Concentration Assay

Protein concentration was determined using a spectrophotometer at afixed absorbance of 280 nm with 1 cm path length of cell. Triplicatereadings were measured for a tested sample with water and buffer ascontrols. Protein concentration was determined using extinctioncoefficient at 0.799 mL/mg protein.

1.10 Formulation of Final Product

The formulation buffer contained pyrogen-free PBS, pH 6.5, 2.5%mannitol, and 0.05% Polysorbate 80 that was degassed by vacuum andsterile filtered (0.2 μm).

Endotoxin was removed using a Detoxi-Gel™ equilibrated with 5 columnbeds of the formulation buffer (PBS, pH 6.5, 2.5% mannitol, and 0.05%Polysorbate 80). The flow rate was controlled by gravity at ˜0.3 mL/min.Product samples were applied onto the gel, and the product eluted usingthe formulation buffer. The volume of the collected product was adjustedwith additional formulation buffer to provide a protein concentration ofabout 100 μg/mL.

The peptide formulations were sterile filtered (0.2μ) and the effluentwas dispensed as 1 mL aliquots into 2.0 mL pyrogen-free vials. Inaddition, aliquots were taken for endotoxin and protein analysis. Allproducts were stored at 4° C.

1.13 Pharmacokinetic Study

The pharmacokinetic analysis was performed using radioiodinated protein.After administration of the labeled interferons by IV tail veininjections into the rats, the clearance rate was measured as thereduction in radioactivity in blood drawn at specific intervals over 72h. Each time point is a measure of at least five rats.

1.14 Results

The reaction rate of GalNAc-T2 was measured at two pH's, a neutral pH(7.4) and a slightly acidic pH (6.2). Glycosylation with GalNAcproceeded successfully at both pH 6.2 and pH 7.4. As can be seen in theMALDI analysis of the reaction progress, the reaction rate was faster atpH 7.4 than at pH 6.2.

GalNAc-T2 and GalNAc were added to interferon alpha-2β quantitatively ateither pH 6.2 or pH 7.4. The reaction was followed by MALDI. During theenzymatic reaction, a new interferon alpha mass ion formed (IFN-alpha-2b19,281 Da and IFN-alpha-2β-GalNAc, 19,485 Da).

The product of the reaction at pH 6.2, IFN-alpha-2b-GalNAc, wassubmitted to analysis to determine the position of substitution of theGalNAc on the protein. Peptide mapping and site occupancy mapping wereused for this purpose. Peptide mapping using TIC of LC-MS/MS and a GluCdigest of IFN-alpha-2b produced a peptide fragment of mass 1018.69.MS/MS peptide amino acid sequencing of the peptide mass ion of 1018.69containing the GalNAc indicated that sugar was attached to T¹⁰⁶.

The sialyl-PEGylation of IFN-alpha-2b-GalNAc was examined usingST6GalNAc-1 and CMP-SA-PEG-20 kilodalton. The reaction ofIFN-alpha-2b-GalNAc produced the PEG-ylated protein, which was visibleby SDS PAGE. In general, the reaction proceeded at 32° C. for 96 h. Thereaction was monitored by SDS PAGE. SDS PAGE indicated that about 70% ofthe IFN-alpha-2b-GalNAc was converted to IFN-alpha-2b-GalNAc-SA-PEG-20kilodalton. The MALDI analysis of the new band indicated a mass ion of41,500 Daltons, the mass of IFN-alpha-2b-GalNAc-SA-PEG-20 kilodalton.

The glycoform of PEG-ylated interferon alpha-2b containing theGalNAc-Gal-SA-PEG structure was also produced. The reaction wasperformed using the conditions described above. The desired product wasdetected by SDS PAGE. A one pot, two-step reaction was used to producethe desired product, beginning with IFN-alpha-2β-GalNAc withcore-1-β3-galactosyltransferase-1, ST3Gal2, UDP-galactose andCMP-SA-PEG-20 kilodalton. The reaction was incubated at 32° C. for 96 h.The reaction was monitored by SDS PAGE. After 24 h, the reaction wasabout 70% complete. The MALDI of the product indicated a mass ion of41,900 Da, which originates from the desiredIFN-alpha-2β-GalNAc-Gal-SA-PEG-20 kilodalton product.

Both glycoforms of the PEG-ylated interferon alpha-2b products werepurified using a two-step process. In the first step, ion-exchangechromatography was performed using SP Sepharose. This procedure removedunreacted PEG materials and provided some separation of other proteins.The ion exchange step was followed by separation on SEC. A Superdex 75column was used to remove remaining smaller proteins including theglycosyltransferases and unPEG-ylated interferon alpha. Both PEG-ylatedglycoforms of interferon alpha were purified to greater than 90% asshown by SDS PAGE).

The antiviral data indicates that PEG-ylated glycoforms A and B retaintheir antiviral effects).

The radioiodinated PEG-ylated proteins were injected into rats via theirtail veins, the AUC for both proteins was 5-7 fold greater un-PEG-ylatedinterferon alpha-2β.

Glycoform A (IFN-alpha-2β-GalNAc-SA-PEG-20 kilodalton) and B(IFN-alpha-2β-GalNAc-Gal-SA-PEG-20 kilodalton) were both bioactive.

Example 2 2.1 Preparation of G-CSF-GalNAc (pH 6.2)

960 μg of G-CSF in 3.2 mL of buffer was concentrated by utrafiltrationusing a UF filter (5 kilodalton) and reconstituted with 1 mL of 25 mMMES buffer (pH 6.2, 0.005% NaN₃). UDP-GalNAc (6 mg, 9.24 mM), GalNAc-T2(40 μL, 0.04 U), and 100 mM MnCl₂ (40 μL, 4 mM) were then added and theresulting solution was incubated at room temperature for 48 hours. After48 hours, MALDI indicated the reaction was complete (shift of the massion from 18800 to 19023 mass units). The reaction mixture was purifiedby HPLC using SEC (Superdex 75 and Superdex 200). The column was elutedusing phosphate buffered saline, pH 4.9 and 0.005% Tween 80. The peakcorresponding to G-CSF-GalNAc was collected and concentrated to about150 μL using a Centricon 5 kilodalton filter and the volume was adjustedto 1 mL using PBS (phosphate buffered saline, pH 4.9 and 0.005% Tween80); protein concentration was 1 mg/mL A₂₈₀).

2.2 Preparation of G-CSF-GalNAc-Gal (pH 6.0)

G-CSF-GalNAc (100 μg) was added to a 100 μL of a solution containing 25mM MES buffer, pH 6.0, 1.5 mM UDP-GalNAc, 10 mM MgCl₂ and 80 mUGalNAc-T2. The CMP-SA-PEG-20 kilodalton (0.5 mg, 0.025 μmole),UDP-galactose 75 μg (0.125 μmole), core-1-Gal-T 20 μL (10 mU) were thenadded and the solution which was slowly rocked at 32° C. for 24 hours.MALDI indicated complete conversion of G-CSF-GalNAc intoG-CCSF-GalNAc-Gal.

2.3 Preparation of G-CSF-GalNAc-SA-PEG-20 kilodalton (C)

2.3a Sequential Process (pH 6.2).

A G-CSF-GalNAc solution containing 1 mg of protein was buffer exchangedinto 25 mM MES buffer (pH 6.2, 0.005% NaN₃) then 5 mg, (0.25 μmole)CMP-SA-PEG (20 kilodalton) was added. Finally, 100 μL, of a 100 mM MnCl₂solution and ST6GalNAc-I (100 μL) were added and the reaction mixturewas rocked slowly at 32° C. Aliquots were taken at time points (24, 48and 72 h) and analyzed by SDS-PAGE. After 24 h, no further reaction wasobserved. The reaction mixture was concentrated by spin filtration (5kilodalton), buffer exchanged against 25 mM NaOAc (pH 4.9) andconcentrated to 1 mL. The product was purified using ion exchange(SP-Sepharose, 25 mM NaOAc, pH 4.9) and SEC (Superdex 75; PBS-pH 7.2,0.005% tween 80, 1 ml/min). The desired fraction was collected,concentrated to 0.5 mL and stored at 4° C.

2.3b One Pot Process using ST6GalNAc-I (pH 6.0)

960 μg of G-CSF protein dissolved in 3.2 mL of product formulationbuffer was concentrated by spin filtration (5 kilodalton) to 0.5 mL andreconstituted in 25 mM MES buffer (pH 6.0, 0.005% NaN₃) to a totalvolume of about 1 mL, or a protein concentration of 1 mg/mL. Followingreconstitution UDP-GalNAc (6 mg, 9.21 μmol), GalNAc-T2 (80 μL, 80 mU),CMP-SA-PEG-20 kilodalton (6 mg, 0,3 μmol) and mouse enzyme ST6GalNAc-I(120 μL)were added. The solution was rocked at 32° C. for 48 hours.Following the reaction the product was purified using standardchromatography conditions on SP-Sepharose and SEC as described above. Atotal of 0.5 mg of protein (A₂₈₀) was obtained, for about a 50% overallyield. The product structure was confirmed by analysis with both MALDIand SDS-PAGE

2.4 Preparation of G-CSF-GalNAc-Gal-SA-PEG-20 Kilodalton (D)

2.4a Starting from G-CSF-GalNAc

UDP-galactose (4 mg, 6.5 μmole), core-1-Gal-T₁ (320 μL, 160 mU),CMP-SA-PEG-20 kilodalton (8 mg, 0.4 μmole), ST3Gal2 (80 μL, 0.07 mU) and80 μL of 100 mM MnCl₂ were directly added to the crude 1.5 mL ofreaction mixture of the G-CSF-GalNAc (1.5 mg) in 25 mM MES buffer (pH6.0) from Example 2.1 (above). The resulting mixture was incubated at32° C. for 60 hours, however, the reaction was complete after 24 h. Thereaction mixture was centrifuged and the solution was concentrated to0.2 mL using ultrafiltration (5 kilodalton) and then redissolved in 25mM NaOAc (pH 4.5) to a final volume of 1 mL. The product was purifiedusing SP-Sepharose, the peak fractions were concentrated using a spinfilter (5 kilodalton) and the residue purified further using SEC(Superdex 75). After concentration using a spin filter (5 kilodalton),the protein was diluted to 1 mL using formulation buffer consisting ofPBS, 2.5% mannitol, 0.005% polysorbate, pH 6.5, and formulated at aprotein concentration of 850 μg protein per mL (A₂₈₀). The overall yieldwas 55%. The MALDI analysis is shown in FIG. 28.

2.4b Starting from G-CSF

960 μg, of G-CSF (3.2 mL) was concentrated by spin filter (5 kilodalton)and reconstituted with 25 mM MES buffer (pH 6.0, 0.005% NaN₃). The totalvolume of the G-CSF solution was adjusted to about 1 mg/mL andUDP-GalNAc (6 mg), GalNAc-T2 (80 μL), UDP-galactose (6 mg),core-1-Gal-T₁ (160 μL, 80 μU), CMP-SA-PEG (20 kilodalton) (6 mg),ST3Gal-2 (160 μL, 120 μU) and MnCl₂ (40 μL of a 100 mM solution) wereadded. The resulting mixture was incubated at 32° C. for 48 h.

2.5 SP Sepharose HPLC Chromatography

The SP Sepharose was performed as described in Example 1.4.

2.6 Size Exclusion Chromatography

SEC was performed as described in Example 1.5. The purified samples werestored at 4° C.

2.6a Hydrophobic Interaction Chromatography (HIC)

Following the first step of chromatographic chromatography HIC can beused as a second purification step to remove contaminants other thenun-Pegylated G-CSF. Thus, a method is available for the purification ofglycopegylated G-CSF that has been through an initial purification on agel permeation column.

2.7 SDS PAGE Analysis

The SDS PAGE was performed as set forth in Example 1.6.

2.8 MALDI Analysis

MALDI analysis was performed as described in Example 1.7.

2.9 Peptide Mapping Analysis

Protein mapping analysis was performed as illustrated in Example 1.8

2.10 Protein Concentration Assay

Protein concentration was determined as described in Example 1.9.

2.11 Product Formulation

The product was formulated as set forth in Example 1.10

2.12 Endotoxin Determination

Endotoxin was determined as set forth in Example 1.11.

2.13 Cell Proliferation Assay

A G-CSF proliferation assay with a NFS-60 cell line and a Tf-1 cell linewere performed according to standard procedures. The cells were platedinto a 96 well plate at 25000 cell/ml in the presence of differentconcentrations of G-CSF (51 nM, 25.5 nM, 12.75 nM, 3.2 nM, 1.6 nM, 0.8nM, 0 nM), a chemically PEG-ylated G-CSF analogue, and PEGylated G-CSF Cfrom Example 2.3 (above), and PEGylated G-CSF D from Example 2.4(above). The cells were incubated at 37° C. for 48 hours. A calorimetricMTT assay was used to determine the cell viability.

2.14 In Vivo Activity: White Blood Cell (WBC) Production in the Rat

Two doses of drug (50 μg/kg, 250 μg/kg) were examined for each of C,G-CSF and a chemically PEG-ylated G-CSF using mice. Blood was drawn attime points of 2 hour, 12 hour, 24 hour, 36 hour, 48 hour, 60 hour, 72hour, 84 hour and 96 hour, and the WBC and neutrophil counts weremeasured (FIG. 4).

2.15 Accelerated Stability Study

An accelerated stability study of PEGylated G-CSF, C, from Example 2.3,and PEGylated G-CSF, D, from Example 2.4 was performed using a buffer atpH 8.0 heated to 40° C. 72 μg of PEGylated G-CSF C, was diluted to 8 mLwith formulation buffer (PBS, 2.5% mannitol, 0.005% polysorbate 80). 1mg of PEGylated G-CSF D, was diluted with 16 mL of formulation buffer.Both solutions were adjusted to pH 8.0 with NaOH and the resultingsolution was sterile filtered into pyrogen-free tubes. The samples wereslowly rotated at 40° C. and aliquots (0.8 mL) were taken at timepointsof 0 hour, 72 hours and 168 hours. Analysis was performed using SEC(Superdex 200) as described above (FIG. 6 and FIG. 7).

2.16 Protein Radiolabeling

G-CSF was radiolabeled using the Bolton Hunter reagent. This reactionwas performed at pH 7.4 for 15 minutes and was followed by a SEC(Superdex 200) purification. Once purified, the formulation buffer pHwas adjusted to 5.0 and the protein concentration was determined byA₂₈₀.

2.17 ELISA Assay

An Elisa assay was utilized to quantify the G-CSF derivatives in ratplasma. The pharmacokinetic results are shown in FIG. 9.

2.18 Pharmacokinetic Study

Two pharmacokinetic studies were performed. For the firstpharmacokinetic study proteins were radiolabeled and administered by IVtail vein injections into rats. Clearance rate was measured as thereduction in radioactivity in blood drawn at specific intervals over 48hours. Each time point was a measure of at least five rats.

Specifically, 10 μg of G-CSF derivative was injected per animal (1 μg oflabeled protein and 9 μg of unlabeled protein). In addition to the bloodbeing drawn and counted as described above, plasma was also collectedand the protein acid was precipitated. The protein pellets were thenalso counted for radioactivity. The data from these studies is shown isFIG. 2, FIG. 3 and FIG. 8.

In the second pharmacokinetic study the unlabeled G-CSF derivatives (30μg per animal) were administered by IV tail vein injections into rats.Blood samples were drawn at the time points indicated and the samplesanalzed by the G-CSF ELISA assay. The data is shown in FIG. 9.

2.19 Results

Human GalNAc T2 transferred GalNAc to G-CSF expressed in E. coli\usingUDP-GalNAc as the donor. Depending on the pH of the reaction buffer, oneor two GalNAc moities were added to G-CSF as determined by MALDI.Addition of the second GalNAc proceeded slowly amounting to about 10-15%of the total product. One GalNAc could be selectively added to G-CSF, inconversion yields of over 90%, by adjusting the pH of the reactionsolution to 6.0-6.2. Addition of the second GalNAc occurred when thereaction was performed at a pH between about 7.2 and 7.4. Both Co+² andMn⁺² are useful divalent metal ions in the reaction. Peptide mapping ofthe reaction products indicated that the predominant product of thereaction was addition of GalNAc to threonine-133, the natural site ofO-linked glycosylation in mammalian systems\. The second GalNAc wasobserved in the amino terminal peptide fragment of G-CSF and ispostulated to occur at threonine-2.

The reaction of G-CSF-GalNAc with ST6GalNAc-1 (chicken or mouse) andCMP-SA-PEG-20 kilodalton provided the product G-CSF-GalNAc-SA-PEG-20kilodalton, which was verified by MALDI, with conversion yields of about50% as determined by SDS-PAGE\. The G-CSF-GalNAc could also be furtherelongated using core-1-Gal-T and UDP-galactose to provide completeconversion to G-CSF-GalNAc-Gal\. Glyco-PEG-ylation of this intermediatewith ST3Gal2 and CMP-SA-PEG-20 kilodalton then provided the productG-CSF-GalNAc-Gal-SA-PEG-20 kilodalton in overall yields of about 50%\.These reactions were performed either sequentially in one pot orsimultaneously in one pot starting from G-CSF or its glycosylatedintermediates. In these studies, little or no difference was observed inoverall yield by using either approach.

The products of the glycosylation or glyco-PEG-ylation reactions werepurified using a combination of ion exchange and SEC. The ion exchangestep removes the unreacted G-CSF or its glycosylated intermediates(GalNAc or GalNAc-Gal) as well as any unreacted CMP-SA-PEG-20kilodalton. The SEC step removed remaining unreacted G-CSF and otherprotein contaminants from the glycosyltransferases used in the process\.The G-CSF's containing the GalNAc-SA-PEG-20 kilodalton or theGalNAc-Gal-SA-PEG-20 kilodalton had identical properties and retentiontimes using these purification methods. The final products had typicalprofiles as shown in.

Once purified, the PEG-ylated proteins were formulated in a PBS buffercontaining 2.5% mannitol and 0.005% Tween 80. Initially, pH 6.5 was usedin the formulation but aggregation of the glyco-PEG-ylated protein was aconcern (see below) so the formulation buffer pH was lowered to 5.0.Literature reports have indicated that G-CSF aggregation is prevented bymaintaining a solution pH between 4-5. Endotoxin was removed using anendotoxin removal cartridge using sterile technique. Proteinconcentrations were typically adjusted to concentrations between 100μg/mL to 1 mg/mL as required for biological studies. Endotoxincalculations were typically below 3 EU/ml by this process. Theformulated products are stored at 4.

The products were tested in an in vitro cell proliferation assay usingNSF-60 cells sensitive to G-CSF. It was observed that both theGalNAc-SA-PEG-20 kilodalton and GalNAc-Gal-SA-20 kilodalton productswere effective at initiating cell proliferation (FIG. 1).

An accelerated stability study was performed on a chemically PEG-ylateG-CSF and C (G-CSF-GalNAc-SA-PEG-20 kilodalton). The formulation bufferpH was adjusted to 8.0 and the temperature was raised to 40° C. Sampleswere taken of each protein at times 0, 72 and 168 h (FIG. 6 and FIG. 7).Chemically PEG-ylated G-CSF was observed to aggregate entirely underthese conditions within 168 h. SEC using a Superdex 200 chromatographywas used to separate the aggregates. Although the glycoconjugateG-CSF-GalNAc-SA-PEG-20 kilodalton also formed aggregates that wereseparable using SEC, the aggregation occurred at a much slower rate.

The glyco-PEG-ylated G-CSF was radioiodinated using the Bolton Hunterreagent. A cold labeling study was also performed prior to the actualradiolabeling to determine the extent of aggregation and to establish amethodology for removing any aggregates formed. Use of the Bolton Hunterreagent (cold) did provide some aggregates as shown in FIG. 5. SEC usinga Superdex 200 column removed the aggregates and provided the monomeric,labeled material. Similar results were obtained using ¹²⁵I labeledreagent. The use of the formulation minimized aggregation on storage.Protein content was measured by measuring the absorbance at A₂₈₀.

The results of the rat pK study incorporating G-CSF, chemicallyPEG-ylated G-CSF and the PEG-G-CSF conjugate labeled with the BoltonHunter reagent are shown in FIG. 3. In this study, blood and proteinprecipitated from plasma were counted for radioactivity after IVadministration of 10 μg of G-CSF conjugate per rat. The data from bothblood and plasma protein clearly indicate that the PEG conjugate andChemically PEG-ylated G-CSF have identical clearance rates (FIG. 3 andFIG. 8).

The ability of the G-CSF derivatives to initiate WBC production was thenexamined in a mouse model. Each test compound was injected IV as asingle bolus and the induction of WBC and neutrophils was monitored overtime. Chemically PEG-ylated G-CSF was the most potent protein testedwhen administered at 250 μg/kg. The PEG conjugate(G-CSF-GalNAc-SA-PEG-20 kilodalton) induced WBC production to almost thesame degree as Chemically PEG-ylated G-CSF at 250 μg/kg, and far greaterthan G-CSF at a similar concentration.

Example 3

This example discloses amino acid sequence mutations that introducechanges introduce O-linked glycosylation sites, i.e., serine orthreonine residues, into a preferably proline-containing site in the 175amino acid wild-type sequence of G-CSF or any modified version thereof.As a reference the 175 amino acid wild-type G-CSF sequence is shownbelow:

(SEQ ID NO:2) MTPLGPASSLP QSFLLKCLEQ VRKIQGDGAA LQEKLCA TYKLCHPEELVLLGHSLGIP WAPLSSCPSQ ALQLAGCLSQ LHSGLFLYQG LLQALEGISP ELGPTLDTLQLDVADFATTI WQQMEELGMA PALQPTQGAM PAFASAFQRR AGGVLVASHL QSFLEVSYRVLRHLAQP

3.1 N-Terminal Mutations

In the N-terminal mutants, the N-terminus of a wild-type G-CSF, M¹TPLGPA(SEQ ID NO:), is replaced with either M¹X_(n)TPLGPA orM¹B_(o)PZ_(m)X_(n)TPLGPA. Wherein n, o and m are integers selected from0 to 3, and at least one of X, B and O is Thr or Ser. When more than oneof X, B and O is Thr or Ser, the identity of these moieties isindependently selected. Where they appear, superscripts denote theposition of the amino acid in the wild-type starting sequence.

Preferred examples include:

M ¹VTPL⁴GPA (SEQ ID NO:) M ¹QTPL⁴GPA (SEQ ID NO:) M ¹ATPL⁴GPA (SEQ IDNO:) M ¹PTQGAMPL⁴GPA (SEQ ID NO:) M ¹VQTPL⁴GPA (SEQ ID NO:) M ¹QSTPL⁴GPA(SEQ ID NO:) M ¹GQTPL⁴GPA (SEQ ID NO:) M ¹APTSSSPL⁴GPA (SEQ ID NO:) M¹APTPL⁴GPA (SEQ ID NO:)

3.2 Internal Mutation Site 1

In these mutants, the N-terminus of a wild-type GCSF, M¹TPLGP (SEQ IDNO:8), is replaced with M¹TPX_(n)B_(o)O_(r)P. Wherein n, o and r areintegers selected from 0 to 3, and at least one of X, B and O is Thr orSer. When more than one of X, B and O is Thr or Ser, the identity ofthese moieties is independently selected. Where they appear,superscripts denote the position of the amino acid in the wild-typestarting sequence.

Preferred mutations include:

M ¹TPTLGP (SEQ ID NO:8) M ¹TPTQLGP (SEQ ID NO:8) M ¹TPTSLGP (SEQ IDNO:8) M ¹TPTQGP (SEQ ID NO:8) M ¹TPTSSP (SEQ ID NO:8) M¹TPQTP (SEQ IDNO:8) M¹TPTGP (SEQ ID NO:8) M¹TPLTP (SEQ ID NO:8) M¹TPNTGP (SEQ ID NO:8)M¹TPVTP (SEQ ID NO:8) M¹TPMVTP (SEQ ID NO:8) MT¹P²TQGL³G⁴P⁵A⁶S⁷ (SEQ IDNO:8)

3.3 Internal Mutation Site 2

This mutation is made for the purpose of maintaining G-CSF activity. Inthese mutants, the amino acid sequence containing H⁵³, LGH⁵³SLGI (SEQ IDNO:) is mutated to LGH⁵³BOLGI, where Θ is H, S, R, E or Y, and B iseither Thr or Ser.

Preferred examples include:

LGHTLGI

LGSSLGI

LGYSLGI

LGESLGI

LGSTLGI

3.4 Internal Mutation Site 3

In this type of mutant, the amino acid sequence encompassing P¹²⁹,P¹²⁹ALQPT (SEQ ID NO:), is mutated to P¹²⁹Z_(m)J_(q)O_(r)X_(n)PT,wherein Z, J, O and X are independently selected from Thr or Ser, and m,q, r, and n are integers selected from 0 to 3.

Preferred examples include:

P¹²⁹TLGPT

P¹²⁹ TQGPT

P¹²⁹TSSPT

P¹²⁹TQGAPT

P¹²⁹ NTGPT

P¹²⁹ALTPT

P¹²⁹ MVTPT

P¹²⁹ASSTPT

P¹²⁹TTQP

P¹²⁹ NTLP

P¹²⁹TLQP

MAP¹²⁹ATQPTQGAM

MP¹²⁹ATTQPTQGAM

3.5 Internal Mutation Site 4

In this type of mutant, the amino acid sequence surrounding P⁶¹,LGIPWAP⁶¹LSSC (SEQ ID NO:), is replaced withPZ_(m)U_(s)J_(q)P⁶¹O_(r)X_(n)B_(o)C, wherein m, s, q, r, n, and o areintegers selected from 0 to 3, and at least one of Z, J, O, X, B and Uis selected as either Thr or Ser. When more than one of Z, J, O, X, Band U is Thr or Ser, each is independently selected

Preferred examples include:

P⁶¹ TSSC

P⁶¹ TSSAC

LGIPTA P⁶¹ LSSC

LGIPTQ P⁶¹TLSSC

LGIPTQG P⁶¹LSSC

LGIPQT P⁶¹PLSSC

LGIPTS P⁶¹LSSC

LGIPTS P⁶¹ LSSC

LGIPTQP⁶¹LSSC

LGTPWAP⁶¹LSSC

LGTPFA P⁶¹LSSC

P⁶¹ FTP

SLGAP⁵⁸TAP⁶¹LSS

3.6 C-Terminal Mutations

In this type of mutant, the amino acid sequence at the C-terminus of awild-type G-CSF, RHLAQP¹⁷⁵ (SEQ ID NO:) is replaced withØ_(a)G_(p)J_(q)O_(r)P¹⁷⁵X_(n)B_(o)Z_(m)U_(s)Ψ_(t), wherein a, p, q, r,n, o, m, s, and t are integers selected from 0 to 3, and at least one ofZ, U, O, J, G, Ø, B and X is Thr or Ser and when more than one of Z, U,O, J, G, Ø, B and X are Thr or Ser, they are independently selected. Øis optionally R, and G is optionally H. The symbol T represents anyuncharged amino acid residue or E (glutamate).

Preferred examples include:

RHLAQTP¹⁷⁵

RHLAGQTP¹⁷⁵

QP¹⁷⁵TQGAMP

RHLAQTP¹⁷⁵AM

QP¹⁷⁵TSSAP

QP¹⁷⁵TSSAP

QP¹⁷⁵TQGAMP

QP¹⁷⁵TQGAM

QP¹⁷⁵TQGA

QP¹⁷⁵TVM

QP¹⁷⁵NTGP

QP¹⁷⁵ QTLP

3.7 Internal Mutations surrounding P¹³³

Additional G-CSF mutants include those with internal mutationssurrounding the amino acid P¹³³. Examples include:

P¹³³TQTAMP¹³⁹

P¹³³TQGTMP

P¹³³TQGTNP

P¹³³TQGTLP

PALQP¹³³TQTAMPA

Example 4

Mutations in the amino acid sequence of granulocyte colony stimulatingfactor (G-CSF) can introduce additional sites for O-linkedglycosylation, such that the protein may be modified at these sitesusing the method of the present invention. This example sets forthselected representative mutants of the invention.

4.1 G-CSF (wild type 178 aa variant) (SEQ ID NO: 1) mtplgpasslpqsfllkcleq vrkiqgdgaa lqeklvseca tyklchpeel vllghslgip waplsscpsqalqlagclsq lhsglflyqg llqalegisp elgptldtlq ldvadfatti wqqmeelgmapalqptqgam pafasafqrr aggvlvashl qsflevsyrv lrhlaqp 4.2 G-CSF (wild type175 aa variant) (SEQ ID NO:3) mtplgpasslp qsfllkcleq vrkiqgdgaa lqeklcaatyklchpeel vllghslgip waplsscpsq alqlagclsq lhsglflyqg llqalegispelgptldtlq ldvadfatti wqqmeelgma palqptqgam pafasafqrr aggvlvashlqsflevsyrv lrhlaqp 4.9 G-CSF Mutant 1 (Amino Terminal mutation)miatplgpasslp qsfllkcleq vrkiqgdgaa lqeklcaatyk lchpeelvll ghslgipwaplsscpsqalq lagclsqlhs glflyqgllq alegispelg ptldtlqldv adfattiwqqmeelgmapal qptqgampaf asafqrragg vlvashlqsf levsyrvlrh laqp 4.10 G-CSFMutant 2 (Amino Terminal mutation) mgvtetplgpasslp qsfllkcleq vrkiqgdgaalqeklcaatyk lchpeelvll ghslgipwap lsscpsqalq lagclsqlhs glflyqgllqalegispelg ptldtlqldv adfattiwqq meelgmapal qptqgampaf asafqrraggvlvashlqsf levsyrvlrh laqp 4.11 G-CSF Mutant 3 (Amino Terminal mutation)maptplgpasslp qsfllkcleq vrkiqgdgaa lqeklcaatyk lchpeelvll ghslgipwaplsscpsqalq lagclsqlhs glflyqgllq alegispelg ptldtlqldv adfattiwqqmeelgmapal qptqgampaf asafqrragg vlvashlqsf levsyrvlrh laqp 4.12 G-CSFMutant 4 (Site 1) mtp ³tqglgpasslp qsfllkcleq vrkiqgdgaa lqeklcaatyklchpeelvll ghslgipwap lsscpsqalq lagclsqlhs glflyqgllq alegispelgptldtlqldv adfattiwqq meelgmapal qptqgampaf asafqrragg vlvashlqsflevsyrvlrh laqp 4.13 G-CSF Mutant 5 (Site 3) Mtplgpasslp qsfllkcleqvrkiqgdgaa lqeklcaatyk lchpeelvll ghslgipwap lsscpsqalq lagclsqlhsglflyqgllq alegispelg ptldtlqldv adfattiwqq meelgmap ¹²⁹at qptqgampafasafqrragg vlvashlqsf levsyrvlrh laqp 4.14 G-CSF Mutant 6 (Site 4)Mtplgpasslp qsfllkcleq vrkiqgdgaa lqeklcaatyk lchpeelvll ghslgip⁵⁸ftplsscpsqalq lagclsqlhs glflyqgllq alegispelg ptldtlqldv adfattiwqqmeelgmapaL qptqgampaf asafqrragg vlvashlqsf levsyrvlrh laqp

Example 5 GlycoPEGylation of G-CSF Produced in CHO Cells

5a. Preparation of Asialo-Granulocyte-Colony Stimulation Factor (G-CSF)

G-CSF produced in CHO cells was dissolved at 2.5 mg/mL in 50 mM Tris 50mM Tris-HCl pH 7.4, 0.15 M NaCl, 5 mM CaCl₂ and concentrated to 500 μLin a Centricon Plus 20 centrifugal filter. The solution was incubatedwith 300 mU/mL Neuraminidase II (Vibrio cholerae) for 16 hours at 32° C.To monitor the reaction a small aliquot of the reaction was diluted withthe appropriate buffer and a IEF gel performed. The reaction mixture wasthen added to prewashed N-(p-aminophenyl)oxamic acid-agarose conjugate(800 μL/mL reaction volume) and the washed beads gently rotated for 24hours at 4° C. The mixture was centrifuged at 10,000 rpm and thesupernatant was collected. The beads were washed 3 times with Tris-EDTAbuffer, once with 0.4 mL Tris-EDTA buffer and once with 0.2 mL of theTris-EDTA buffer and all supernatants were pooled. The supernatant wasdialyzed at 4° C. against 50 mM Tris-HCl pH 7.4, 1 M NaCl, 0.05% NaN₃and then twice more against 50 mM Tris-HCl pH 7.4, 1 M NaCl, 0.05% NaN₃.The dialyzed solution was then concentrated using a Centricon Plus 20centrifugal filter and stored at −20° C. The conditions for the IEF gelwere run according to the procedures and reagents provided byInvitrogen. Samples of native and desialylated G-CSF were dialyzedagainst water and analyzed by MALDI-TOF MS.

5b. Preparation of G-CSF-(alpha-2,3)-Sialyl-PEG

Desialylated G-CSF was dissolved at 2.5 mg/mL in 50 mM Tris-HCl, 0.15 MNaCl, 0.05% NaN₃, pH 7.2. The solution was incubated with 1 mMCMP-sialic acid-PEG and 0.1 U/mL of ST3Gall at 32° C. for 2 days. Tomonitor the incorporation of sialic acid-PEG, a small aliquot of thereaction had CMP-SA-PEG-fluorescent ligand added; the label incorporatedinto the peptide was separated from the free label by gel filtration ona Toso Haas G3000SW analytical column using PBS buffer (pH 7.1). Thefluorescent label incorporation into the peptide was quantitated usingan in-line fluorescent detector. After 2 days, the reaction mixture waspurified using a Toso Haas G3000SW preparative column using PBS buffer(pH 7.1) and collecting fractions based on UV absorption. The product ofthe reaction was analyzed using SDS-PAGE and IEF analysis according tothe procedures and reagents supplied by Invitrogen. Samples of nativeand PEGylated G-CSF were dialyzed against water and analyzed byMALDI-TOF MS.

5c. Preparation of G-CSF-(alpha-2,8)-Sialyl-PEG

G-CSF produced in CHO cells, which contains an alpha 2,3-sialylatedO-linked glycan, was dissolved at 2.5 mg/mL in 50 mM Tris-HCl, 0.15 MNaCl, 0.05% NaN₃, pH 7.2. The solution was incubated with 1 mMCMP-sialic acid-PEG and 0.1 U/mL of CST-II at 32° C. for 2 days. Tomonitor the incorporation of sialic acid-PEG, a small aliquot of thereaction has CMP-SA-PEG-fluorescent ligand added; the label incorporatedinto the peptide was separated from the free label by gel filtration ona Toso Haas G3000SW analytical column using PBS buffer (pH 7.1). Thefluorescent label incorporation into the peptide was quantitated usingan in-line fluorescent detector. After 2 days, the reaction mixture waspurified using a Toso Haas G3000SW preparative column using PBS buffer(pH 7.1) and collecting fractions based on UV absorption. The product ofthe reaction was analyzed using SDS-PAGE and IEF analysis according tothe procedures and reagents supplied by invitrogen. Samples of nativeand PEGylated G-CSF were dialyzed against water and analyzed byMALDI-TOF MS.

5d. Preparation of G-CSF-(alpha 2,6)-Sialyl-PEG

G-CSF, containing only O-linked GalNAc, was dissolved at 2.5 mg/mL in 50mM Tris-HCl, 0.15 M NaCl, 0.05% NaN₃, pH 7.2. The solution was incubatedwith 1 mM CMP-sialic acid-PEG and 0.1 U/mL of ST6GalNAcI or II at 32° C.for 2 days. To monitor the incorporation of sialic acid-PEG, a smallaliquot of the reaction has CMP-SA-PEG-fluorescent ligand added; thelabel incorporated into the peptide was separated from the free label bygel filtration on a Toso Haas G3000SW analytical column using PBS buffer(pH 7.1). The fluorescent label incorporation into the peptide wasquantitated using an in-line fluorescent detector. After 2 days, thereaction mixture was purified using a Toso Haas G3000SW preparativecolumn using PBS buffer (pH 7.1) and collecting fractions based on UVabsorption. The product of the reaction was analyzed using SDS-PAGE andIEF analysis according to the procedures and reagents supplied byInvitrogen. Samples of native and PEGylated G-CSF were dialyzed againstwater and analyzed by MALDI-TOF MS.

G-CSF produced in CHO cells was treated with Arthrobacter sialidase andwas then purified by size exclusion on Superdex 75 and was treated withST3Gall or ST3 Gal2 and then with CMP-SA-PEG 20 Kda. The resultingmolecule was purified by ion exchange and gel filtration and analysis bySDS PAGE demonstrated that the PEGylation was complete. This was thefirst demonstration of glycoPEGylation of an O-linked glycan.

Example 6 Recombinant GCSF—Expression, Refolding and Purification

-   -   Harvest cells by centrifugation, discard supernatant. Results of        growth on various media are shown in FIG. 9.    -   Resuspend cell pellet in 10 mM Tris pH7.4, 75 mM NaCl, 5 mM        EDTA—use 10 ml/g (lysis buffer)    -   Microlluidize cells (French press works as well)    -   Centrifuge 30 min, 4° C. at 5,000 RPM-discard supernatant    -   Resuspend pellet in lysis buffer and centrifuge as above    -   Wash IB's in 25 mM Tris pH8, 100 mM NaCl, 1% TX-100, 1% NaDOC, 5        mM EDTA. Pellets are resuspended by pipetting and vortexing.        Centrifuge 15 min 4° C. 5,000 RPM. Repeat this step once more        (total of two washes)    -   Wash pellets two times in 25 mM Tris pH8, 100 mM NaCl, 5 mM EDTA        to remove detergents, centrifuge as above    -   Resuspend pellets in dH₂O to aliquot and centrifuge as above.        Pellets are frozen at −2OC    -   IB's are resuspended at 20 mg/ml in 6M guanidine HCl, 5 mM EDTA,        100 mM NaCl, 100 mM Tris pH8, 10 mM DTT using a pipettor,        followed by rotation for 2-4 h at room temperature.    -   Centrifuge solubilized IB's for 1 min at room temperature at        14,000 RPM. Save supernatant.    -   Dilute supernatant 1:20 with refold buffer 50 mM MES pH6, 240 mM        NaCl, 10 mM    -   KC1, 0.3 mM lauryl maltoside, 0.055% PEG3350, 1 mM GSH, 0.1 M        GSSG, 0.5M arginine and refold on rotator overnight at 4° C.    -   Transfer refold to Pierce snakeskin 7 kDa MWCO for dialysis.        Dialysis buffer 20 mM NaOAc pH4, 50 mM NaCI, 0.005% Tween-80,        0.1 mM EDTA. Dialyze a total of 3 times versus at least a 200        fold excess at 4° C.    -   After dialysis pass material through a 0.45 μM filter.    -   Equlibrate SP-sepharose column with the dialysis buffer and        apply sample. Wash column with dialysis buffer and elute with        dialysis buffer containing a salt gradient up to 1 M NaCl.        Protein typically is eluted at 300-400 mM NaCl.    -   Check material on SDS-PAGE (see e.g., FIG. 10).

Example 7 The Two Enzyme Method in Two Pots

The following example illustrates the preparation of G-CSF-GalNAc-SA-PEGin two sequential steps wherein each intermediate product is purifiedbefore it is used in the next step.

7a. Preparation of G-CSF-GalNAc (pH 6.2) from G-CSF and UDP-GalNAc usingGalNAc-T2.

G-CSF (960 μg) in 3.2 mL of packaged buffer was concentrated byutrafiltration using an UF filter (MWCO 5K) and then reconstituted with1 mL of 25 mM MES buffer (pH 6.2, 0.005% NaN₃). UDP-GalNAc (6 mg, 9.24mM), GalNAc-T2 (40 μL, 0.04 U), and 100 mM MnCl₂ (40 μL, 4 mM) were thenadded and the resulting solution was incubated at room temperature.

After 24 hrs, MALDI indicated the reaction was complete. The reactionmixture was directly subjected to HPLC purification using SEC (Superdex75 and Superdex 200) and an elution buffer comprising of PBS (phosphatebuffered saline, pH 4.9 and 0.005% Tween 80). The collected peak ofG-CSF-GalNAc was concentrated using a Centricon 5 KDa MWCO filter toabout 150 μL and the volume adjusted to 1 ml using PBS (phosphatebuffered saline, pH 4.9 and 0.005% Tween 80). Final proteinconcentration 1 mg/mL (A₂₈₀), yield 100%. The sample was stored at 4° C.

7b. Preparation of G-CSF-GalNAc-SA-PEG using Purified G-CSF-GalNAc,CMP-SA-PEG (20 KDa) and Mouse ST6GalNAc-TI (pH 6.2).

The G-CSF-GalNAc solution containing 1 mg of protein was bufferexchanged into 25 mM MES buffer (pH 6.2, 0.005% NaN₃) and CMP-SA-PEG (20KDa) (5 mg, 0.25 umol) was added. After dissolving, MnCl₂ (100 mcL, 100mM solution) and ST6GalNAc-I (100 mcL, mouse enzyme) was added and thereaction mixture rocked slowly at 32° C. for three days. The reactionmixture was concentrated by ultrafiltration (MWCO 5K) and bufferexchanged with 25 mM NaOAc (pH 4.9) one time and then concentrated to 1mL of total volume. The product was then purified using SP-sepharose (A:25 mM NaOAc+0.005% tween-80 pH 4.5; B: 25 mM NaOAc+0.005% tween-80 pH4.5+2M NaCl) at retention time 13-18 mins and SEC (Superdex 75; PBS-pH7.2, 0.005% Tween 80) at retention time 8.6 mins (superdex 75, flow 1ml/min) The desired fractions were collected, concentrated to 0.5 mL andstored at 4° C.

Example 8 One Pot Method to Make G-CSF-GalNAc-SA-PEG with SimultaneousAddition of Enzymes

The following example illustrates the preparation of G-CSF-GalNAc-SA-PEGin one pot using simultaneous addition of enzymes

8a. One Pot Process Using Mouse ST6GalNAc-I (pH 6.0).

G-CSF (960 μg of protein dissolved in 3.2 mL of the product formulationbuffer) was concentrated by ultrafiltration (MWCO 5K) to 0.5 ml andreconstituted with 25 mM MES buffer (pH 6.0, 0.005% NaN₃) to a totalvolume of about 1 mL or a protein concentration of 1 mg/mL. UDP-GalNAc(6 mg, 9.21 μmol), GalNAc-T2 (80 μL, 80 mU), CMP-SA-PEG (20 KDa) (6 mg,0.3 μmol) and mouse enzyme ST6GalNAc-I (120 μL) and 100 mM MnCl₂ (50 μL)were then added. The solution was rocked at 32° C. for 48 hrs andpurified using standard chromatography conditions on SP-sepharose. Atotal of 0.5 mg of protein (A₂₈₀) was obtained or about a 50% overallyield. The product structure was confirmed by analysis with both MALDIand SDS-PAGE.

8b. One Pot Process Using Chicken ST6GalNAc-I (pH 6.0).

14.4 mg of G-CSF; was concentrated to 3 mL final volume, bufferexchanged with 25 mM MES buffer (pH 6.0, 0.05% NaN₃, 0.004% Tween 80)and the volume was adjusted to 13 mL. The UDP-GalNAc (90 mg, 150 μmole),GalNAc-T2 (0.59 U), CMP-SA-PEG-20 KDa (90 mg), chicken ST6GalNAc-I (0.44U), and 100 mM MnCl₂ (600 mcL) were then added. The resulting mixturestood at room temperature for 60 hrs. The reaction mixture was thenconcentrated using a UF (MWCO 5K) and centrifugation. The residue (about2 mL) was dissolved in 25 mM NaOAc buffer (pH 4.5) and concentratedagain to 5 mL final volume. This sample was purified using SP-sepharosefor about 10-23 min, SEC (Superdex 75, 17 min, flow rate 0.5 ml/min) andan additional SEC (Superdex 200, 23 min, flow rate 0.5 ml/min), to yield3.6 mg (25% overall yield) of G-CSF-GalNAc-SA-PEG-20 KDa (A₂₈₀ and BCAmethod).

Example 9 One Pot Method to Make G-CSF-GalNAc-Gal-SA-PEG with SequentialAddition of Enzymes

The following example illustrates a method for makingG-CSF-GalNAc-Gal-SA-PEG in one pot with sequential addition of enzymes.

9.1 Starting from GalNAc-G-CSF

a. Preparation of G-CSF-GalNAc (pH 6.2) from G-CSF and UDP-GalNAc usingGalNAc-T2.

G-CSF (960 mcg) in 3.2 mL of packaged buffer was concentrated byutrafiltration using an UF filter (MWCO 5K) and then reconstituted with1 mL of 25 mM MES buffer (pH 6.2, 0.005% NaN₃). UDP-GalNAc (6 mg, 9.24mM), GalNAc-T2 (40 μL, 0.04 U), and 100 mM MnCl₂ (40 μL, 4 mM) were thenadded and the resulting solution was incubated at room temperature.

b. Preparation of G-CSF-GalNAc-Gal-SA-PEG from G-CSF-GalNAc;UDP-Galactose, SA-PEG-20 K dalton, and the Appropriate Enzymes

The UDP-Galactose (4 mg, 6.5 μmoles), core-1-Gal-T (320 μL, 160 mU),CMP-SA-PEG-20 KDa (8 mg, 0.4 μmole), ST3Gal2 (80 μL, 0.07 mU) and 100 mMMnCl₂ (80 μL) were directly added to the crude reaction mixture of theG-CSF-GalNAc (1.5 mg) in 1.5 ml 25 mM MES buffer (pH 6.0) from step a,above. The resulting mixture was incubated at 32° C. for 60 hrs. Thereaction mixture was centrifuged and the solution was concentrated usingultrafiltration (MWCO 5K) to 0.2 mL, and then redissolved with 25 mMNaOAc (pH 4.5) to a final volume of 1 mL. The product was purified usingSP-sepharose (retention time of between 10-15 min), the peak fractionwere concentrated using a spin filter (MWCO 5K) and the residue purifiedfurther using SEC (Superdex 75, retention time of 10.2 min). Afterconcentration using a spin filter (MWCO 5K), the protein was diluted to1 mL using formulation buffer with PBS, 2.5% mannitol, 0.005%polysorbate, pH 6.5 and formulated at a protein concentration of 850 mcgprotein per mL (A₂₈₀). The overall yield was 55%.

Example 10 One Pot Method to Make G-CSF-GalNAc-Gal-SA-PEG withSimultaneous Addition of Enzymes

a. Starting from G-CSF.

G-CSF (960 mcg, 3.2 ml) was concentrated by ultrafiltration (MWCO 5K)and reconstituted with 25 mM Mes buffer (pH 6.0, 0.005% NaN₃). The totalvolume of the G-CSF solution was about 1 mg/ml. UDP-GalNAc (6 mg),GalNAc-T2 (80 μL, 80 μU), UDP-Gal (6 mg), Corel GalT (160 μL, ˜80 μU),CMP-SA-PEG(20K) (6 mg) and a 2,3-(O)-sialyltransferase (160 μL, 120 μU),100 mM MnCl₂ (40 μL) were added. The resulting mixture was incubated at32° C. for 48 h. Purification was performed as described below using IEXand SEC. The resulting fraction containing the product were concentratedusing ultrafiltration (MWCO 5K) and the volume was adjusted to about 1mL with buffer. The protein concentration was determined to be 0.392mg/ml by A280, giving an overall yield of 40% from G-CSF.

Example 11

The following Example illustrates an alternative enzymatic method toobtain large quantities of GlycoPEGylated G-CSF.

Granulocyte Colony Stimulating Factor (G-CSF) protein was expressed inE. coli and refolded from inclusion bodies as disclosed in Example X(above).

11a. Priming the Reaction by Addition of GalNAc:

GalNAc-ylation of G-CSF was carried out at 33° C. in 50 mM Bis-Tris pH6.5 buffer containing 1 mM MnCl₂ using refolded GalNAcT2 in the presenceUDP-GalNAc. This step primes the reaction enabling both GalNActransferase and sialyltransferase to work together in subsequent stepsto very efficiently produce maximum amount of GCSF-PEG in a short periodof time.

11b. PEGylation Process:

PEGylation was started 2 (+/−1) hour after GalNAc-ylation by directlyadding CMP-SA-PEG (20K) and ST6GalNAcI (chicken or human) to the primingreaction. This step produces substrate (GCSF-O-GalNAc) for thesialyltransferases to drive the reaction faster in a shorter period oftime than can be achieved in a two step reaction wherein theGCSF-O-GalNAc is first purified from the UDP-GalNAc and other reactioncomponents (see e.g., Example X, above). Furthermore the primed one potreaction produces a higher yield of product than does a one pot reactionin which all components are added simultaneously.

Indeed, comparison of several types of one pot reactions shows that whenall the components were added simultaneously and incubated for 23 hours,the GCSF-PEG produced was 77%. In contrast, when addition of all theenzymes required for the PEGylation reaction was preceded by the 2 hrGalNAc-ylation step described above, product yield was 85%. Therefore,the sequential addition of reaction components resulted in a 10% higheryield than was obtained when all reaction components are addedsimultaneously.

Example 12

This Example describes the results of O-linked GalNAc-ylation of sixmutant G-CSF proteins.

12.1. GalN Acsylation of mutant G-CSF Protein

All the sequences of mutant G-CSF proteins are listed below. Havingthese proteins, O-linked glycosylation was examined. Under the samecondition for glycosylation of native G-CSF, GalNAc-T₂ (BV) was used invitro with UDP-GalNAc in 25 mM MES buffer (pH 6.0). MALDI was used tomonitor the reaction. Measurement of increasing molecular weight ofproteins provided GalNAc addition number. For one addition of GalNAc,increased molecular weight should be 203 Da. Based on MALDI results, wefound that mutant G-CSF-2, -3, -4, accepted one GalNAc; and mutantG-CSF-5 some addition was also observed, and mutant G-CSF-1 accepted twoGalNAcs, forming MAPT-G-CSF(GalNAc)₂ (Molecular weight increasing from18965 to 19369 Daltons).

TABLE X GalNAc addition of Mutant G-CSF (MW measured by MALDI) MW(Intact MW (GalNAc- Number of GalNAc Peptide material) adduct) additionMutantG-CSF-1 18965 19369 2 (MAPT-G-CSF) MutantG-CSF-2 18766 19029 1MutantG-CSF-3 18822 19026 1 MutantG-CSF-4 19369 19574 1 MutantG-CSF-518957 18853 1 MutantG-CSF-6 NT Native G-CSF 18800 19023 1

Peptide mapping and N-terminal analysis were used for determination ofglycosylation sites of MAPT-G-CSF-(GalNAc)₂. In the Glu C-digestedpeptide mapping a G-l+GalNAc peak was found, indicating one GalNAc wasadded at G-1 sequence. N-terminal Edman degradation analysis suggestedthe normal T was lost indicting that GalNAc was added onto T residue.

12.2 GlycoPEGylation of Mutant G-CSF Sequences

a. GlycoPEGylation of Mutant G-CSF Sequence and Buffer Impact on theGlycoPEGylation of MAPT-G-CSF

An examination of glycoPEGylation (20K) of 5 mutants was undertaken.GlycoPEGylation was performed using three enzyme/three nucleotidessystem. (UDP-GalNAc/GalNAc-T₂/UDP-Gal/CoreGalT/CMP-SA-PEG/O-sialyltransferase) in 25 mM MES buffer (pH 6.0). Allmutants can be monoglycoPEGylated. No appreciable diPEGYlation in thiscondition was detected by SDS-PAGE gel by Comassie Blue Stain.

Since MAPT-G-CSF accept two GalNAcs, this mutant should receive two PEGsin theory. Accordingly, we examined the buffer impact on the PEGylationof MAPT-G-CSF as a starting material. Four different buffers (1. 1 M MESbuffer; 2. 25 mM MES buffer (pH 6.0); 3. 50 mM Bis-tris buffer (pH 6.0);4. 1 M HEPS buffer (pH 7.4) were investigated for this reaction. It wasfound that MAPT-G-CSF can be PEGylated in all of the buffer systemtested. However, monoPEGylation product was still a major one. In case 1M MES and 1 M HEPS buffer were used, some diPEGYylation product wasformed, indicating that high concentration buffer improves theglycoPEGylation.

b. Comparision of GlycoPEGylation Efficiency by FormingMAPT-G-CSF(GalNAc-SA-PEG)₂ and MAPT-G-CSF(GalNAc-Gal-SA-PEG)₂

In order to see glycoPEGylation efficiency of Muant G-CSF-1 catalyzed bydifferent enzymes, two enzymes (St₆GalNAcI and O-siayltransferase) wereexamined for sialylPEGylation. Accordingly, MAPT-G-CSF was convertedinto MAPT-G-CSF(GalNAc)₂ and MAPT-G-CSF(GalNAc-Gal)₂ forsiaylPEGylation. The former was treated withCMP-SA-lys-PEG(20K)/St6GalNAc I and the latter was treated withCMP-SA-PEG(20K)/O-sialyltransferase. Both reactions were performed in 25mM MES buffer (pH 6.0) and 1 mg/ml protein concentration. The PEGylationefficiency can be seen in SDS-Page gel. It appeared that two enzymeswere pretty similar in glycoPEGylation of this protein usingCMP-SA-Lys-PEG (20 KDa) under the condition tested.

c. High Protein Concentration Led to Formation ofMAPT-G-CSF((GalNAc-SA-PEG(20 KDa))₂ as a Major Product.

After examining the impact of enzyme and buffer on glycoPEGylation, asdescribed above, the influence of protein concentration on thePEGylation by combining with a factor of high buffer concentration usingST₆GalNAcI as GlycoPEGylation enzyme. So we applied UDP-GalNAc/GalNAc-T₂and CMP-SA-PEG(20 KDa)/St6GalNAcI for glycoPEGylation of MAPT-G-CSFusing 810 mg/ml protein concentration for reaction in 1 M MES buffer (pH6.0). The result suggested that under this condition, the desireddiPEGylation product became the major. Over 90% conversion was alsoachieved by applying more CMP-SA-PEG (20K) and enzyme. PEGylated G-CSFproduct, MAPT-G-CSF((GalNAc-SA-PEG(20 KDa))₂ was purified by combiningSP-Sepharose and SEC purification on Supderdex 200.

12.3. Cell Proliferation Activity of MAPT-G-CSF-(GalNAc-SA-PEG)₂

Cell proliferation assay of MAPT-G-CSF-(GalNAc-SA-PEG)₂ with NFS-60 cellline and Tf-1 cell line were performed. The assay was performed usingprotein concentration between 0 ng/ml to 1000 ng/ml.MAPT-G-CSF(GalNAc-SA-PEG(20K))₂ was active in this assay.

12.4 Experimental Details

12.4a General Procedure of GalNAcsylation of Mutant G-CSF

Certain volume of mutant G-CSF solution (for 100 ug protein) was bufferexchanged with MES buffer (25 mM+0.005% NaN₃, pH 6.0). The final volumewas adjusted to 100 ug/100 ul. To this solution was added 5 ul 100 mMMnCl₂ and GalNAc-T₂ (1 mU). The resulting mixture was rocked at rt for aperiod of time required for MALDI or QTOF analysis.

12.4b Preparation of MAPTP-G-CSF-(GalNAc)₂)

MAPTP-G-CSF 5.4 mg (KJ-675-159, 0.18 mg/ml, 0.053 umol) was exchangedwith MES buffer (25 mM+0.005% NaN₃, pH 6.0). The final volume wasadjusted to 5.4 ml. To this solution, UDP-GalNAc (5 mg, 0.15 umol), 100mM MnCl₂ 0.25 ml and GalNAc-T₂ (1.0 U/ml, 50 ul) were added. Theresulting mixture was rocked at 32° C. for 24 h. M+(MALDI): 19364(MAPT-G-CSF-(GalNAc)₂ verse 18951 (MVPTP-G-CSF).

12.4c General Procedure of GlycoPEGylation of Mutant G-CSF Sequences byOne-Pot Reaction)

Mutant G-CSF 100 ug (Mutant G-CSF-1,2,3,4,5) was mixted with UDP-GalNAc(0.6 mg, 0.923 umol), GalNAc-T₂ (20 ul, 8 mU), UDP-Gal (0.6 mg, 0.923umol), Core 1 Gal T (20 ul, 10 mU), CMP-SA-PEG(20K) (1 mg, 0.05 umol),St3GalIII (20 ul, 28 mU), 100 mM MnCl₂ 3 ul in 100 ul 25 mM MES buffer(pH 6.0+0.005% NaN₃). The resulting mixture was rocked at rt for 24 h.GlycoPEGylation was followed by SDS-PAGE.

12.4d Comparison of Mutant G-CSF-1 GlycoPEGyaltion(20 KDa) in VariousBuffer System

GalNAc₂-MATP-G-CSF (54 ug) was buffer exchanged to the following fourbuffer system (1. 1 M MES buffer (pH 6.0); 2. 25 mM MES buffer (pH 6.0);3. 50 mM Bis-Tris buffer (pH 6.5); 4. 1 M HEPS buffer (pH 7.4). ThenCMP-SA-PEG (20K) (216 ug) ST6GalNAcI (BV, IU/mL, 2.5 ul), 100 mM MnCl₂2.5 ul were added. The resulting mixture was rocked at rt for 24 h.SDS-PAGE gel was used to follow the reaction.

12.4e Comparison of GlycoPEGylation of MAPT-G-CSF by Using ST₆GalNAc Iand O-sialyltransferase (Wang 787-29 and 787-40)

12.4e1 Using St6GalNAc I

First step: 30 ml KJ-675-159 solution (0.18 mg/ml, 5.4 mg protein intotal) was concentrated by ultrifiltration (MWCO 5K) at 3500 g, and thenbuffer exchanged with 25 mM MES buffer (pH 6.0). Final volume wasadjusted to 5.4 ml in a plastic tube. GalNAc-T₂ (1.0 U/ml, 50 ul) wasadded, followed by addition of 0.25 mL MnCl₂. The resulting mixture wasrocked at 32° C. for 24 h. MALDI suggested that the reaction went tocompletion. The reaction mixture was concentrated by UF(MWCO 5K) anddiluted with 25 mM MES buffer to 5 ml, then CMP-SA-PEG(20K) (2×25 mg),ST₆GalNAc_(I) (BV, 1 U/ml), 100 mM MnCl₂ 0.25 ml were added. Theresulting mixture was rocked at 32° C. overnight. SDS-PAGE was used forthe reaction.

12.4e2 Using O-silyltransferase (St₃Gal_(II)):

200 ug GalNAc₂-MATP-G-CSF in 200 ul 25 mM MES buffer (pH 6.0) was mixedwith UDP-Gal 0.6 mg and core GalT (0.2 U/ml, 10 ul) and 10 ul 100 mMMnCl₂. The resulting mixture was rocked at 32° C. for 24 h. The reactionmixture was concentrated by UF (MWCO 5K) and diluted with 25 mM MESbuffer to 200 ul. CMP-SA-PEG (800 ug), St₃GalIII (1.0 U/ml, 10 ul), 10ul 100 mM MnCl₂ were added. The resulting mixture was rocked at rt for24 h. The resulting mixture was rocked at 32° C. overnight. SDS-PAGE gelwas used to follow the reaction.

12.4f MAPTP-G-CSF-(GalNAc-SA-PEG(20K)₂ from glycoPEGylation ofMAPT-G-CSF-(GalNAc)₂ (Wang 787-42)

MAPTP-G-CSF solution (540 ug) was concentrated and exchanged with 1 MMES buffer (pH 6.0) and adjusted to 50 ul. Then UDP-GalNAc (100 ug, 0.15umol, 5 eq), GalNAcT₂ (5.0 U/ml, 5 ul) and 100 mM MnCl₂ (5 ul) wasadded. The resulting mixture was rocked at RT overnight. Then CMP-SA-PEG(20K) (2.16 mg, 0.108 umol) and St₆GaiNAcI (1.0 U/ml, 50 ul) were added.The solution was rocked at rt for 60 h. Additional CMP-SA-PEG(20K) (2.16mg, 0.108 umol) and St6GalNAcI (1.0 U/ml, 50 ul) were added, followed byslow rotation at rt for 24 h. Reaction mixture was exchanged with bufferA (25 mM NaOAc, 0.005% polysorbate 80, pH 4.5), then purified on anAmersham SP-FF (5 mL) column with an isocratic elution of 100% A for 10minutes followed by a linear gradient of 100% A to 20% B over 20 minutesat a flow rate of 3 mL min⁻¹, where B=25 mM NaOAc, 2 M NaCl 0.005%polysorbate 80, pH 4.5. The peak at retention time 17 mins was pooledand concentrated to 0.5 ml, which was further purified on an AmershamHiLoad Superdex 200 (16×600 mm, 34 μm) with phosphate buffered saline,pH 5.0, 0.005% Tween80, at a flow rate of 0.4 mL min⁻¹. Productfractions at retention time 160 mins was pooled, concentrated to provide30 ug of MAPT-G-CSF(GalNAc-SA-PEG(20K))₂(BCA). The yield was notoptimized.

12.4 g Sequences of G-CSF Mutants

Mutant G-CSF-1: (SEQ ID NO: 9)MAPTPLGPASSLPQSFLLKCLEQVRKIQGDGAALQEKLCATYKLCHPEELVLLGHSLGIPWAPLSSCPSQALQLAGCLSQLHSGLFLYQGLLQALEGISPELGPTLDTLQLDVADFATTIWQQMEELGMAPALQPTQGAMPAFASAFQRRAGGVLVASHLQSFLEVSYRVLRHLAQP Mutant G-CSF-2: (SEQ ID NO: )MTPLGPASSLPQSFLLKCLEQVRKIQGDGAALQEKILCATYKILCHPEELVLLGHSLGIPWAPLSSCPSQALQLAGCLSQLHSGLFLYQGLLQALEGISPELGPTLDTLQLDVADFATTIWQQMEELGMAPATQPTQGAMPAFASAFQRRAGGVLVASHLQSFLEVSYRVLRHLAQP Mutant G-CSF-3: (SEQ ID NO: )MTPLGPASSLPQSFLLKCLEQVRKIQGDGAALQEKILCATYKILCHPEELVLLGHSLGIPWAPLSSCPSQALQLAGCLSQLHSGLFLYQGLLQALEGISPELGPTLDTLQLDVADFATTIWQQMEELGMAPALQPTQTAMPAFASAFQRRAGGVLVASHLQSFLEVSYRVLRHLAQP Mutant G-CSF-4 (C-terminal tag): (SEQ IDNO:8) MTPLGPASSLPQSFLLKCLEQVRKIQGDGAALQEKILCATYKILCHPEELVLLGHSLGIPWAPLSSCPSQALQLAGCLSQLHSGLFLYQGLLQALEGISPELGPTLDTLQLDVADFATTIWQQMEELGMAPALQPTQGAMPAFASAFQRRAGGVLVASHLQSFLEVSYRVLRHLAQPTQGAMP Mutant G-CSF-5 (N-terminal MIATP):(SEQ ID NO:10) MIATPLGPASSLPQSFLLKCLEQVRKIQGDGAALQEKILCATYKILCHPEELVLLGHSLGIPWAPLSSCPSQALQLAGCLSQLHSGLFLYQGLLQALEGISPELGPTLDTLQLDVADFATTIWQQMEELGMAPALQPTQGAMPAFASAFQRRAGGVLVASHLQSFLEVSYRVLRHLAQP Mutant G-CSF-6 (177 Mer): (SEQ ID NO:1)MTPLGPASSLPQSFLLKCLEQVRKIQGDGAALQEKILVSECATYKICHPEELVLLGHSLGIPWAPLSSCPSQALQLAGCLSQLHSGLFLYQGLLQALEGISPELGPTLDTLQLDVADFATTIWQQMEELGMAPALQPTQGAMPAFASAFQRRAGGVLVASHLQSFLEVSYRVLRHLAQP Human recombinant G-CSF expressed in Ecoli: (SEQ ID NO:2) MTPLGPASSLPQSFLLKCLEQVRKIQGDGAALQEKILCATYKILCHPEELVLLGHSLGIPWAPLSSCPSQALQLAGCLSQLHSGLFLYQGLLQALEGISPELGPTLDTLQLDYADFATTIWQQMEELGMAPALQPT¹³⁴QGAMPAFASAFQRRAGGVLVASHLQSFLEVSYRVLRHLAQP

Example 13

The following Example illustrates preparation of a GlycoPEGylated hGHprotein The wild-type hGH has no natural glycosylation site, therefore ade novo O-glycosylation site was engineered into a mutant hGH proteinwhich was then be glycosylated with a GalNAc transferase andsialylPEGylated at the mutant site. Five mutant hGH proteins weredesigned to incorporate an O-glycosylation site at either the aminoterminus or in the loop region of the protein molecule. The five mutantproteins were produced and each was tested for hGH activity in a Nb2-11cell proliferation assay.

13.1 Mutant hGH Amino Acid Sequences

192 amino acid Wild-type pituitary derived hGH comprising an N-Terminalmethionine (SEQ ID NO:)MFPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSITNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF 191 amino acid Wild-typepituitary derived hGH lacking an N-Terminal methionine (SEQ ID NO:)FPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSITNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF MVTP mutant: (SEQ ID NO: )(M)VTPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF PTOGAMP mutant: (SEQ ID NO:) MFPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRJSLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPTQGAMPKQTYSKFDTNSITNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF TTT mutant: (SEQ ID NO: )MFPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRJSLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPTTTQIFKQTYSKFDTNSITNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF MAPT mutant: (SEQ ID NO: )MAPTSSPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRJSLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF NTG mutant:MFPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRJSLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPNTGQIFKQTYSKFDTNSITNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF

The four hGH mutants were tested for the ability to act as substratesfor glycosyltransferase GalNAcT2. Of the four hGH mutants, two werefound to be glycosylated by GalNAcT2 by MALDI-MS analysis.

13.2 Preparation of hGH-(TTT)-GalNAc-SA-PEG-30 KDa

For the TTT mutant, GalNAc addition gave rise to a complex mixture ofunglycosylated, and 1-GalNAc and 2-GalNAc species. Peptide mappingexperiments (trypsin digest) showed that the two GalNAc's were added tothe T12 peptide (L129-K141) containing the TTT mutation. The (M)VTPmutant showed only a trace of GalNAc added by MALDI-MS.

The hGH-TTT-mutant (4.0 mL, 6.0 mg, 0.27 micromoles) was bufferexchanged twice with 15 mL of Washing Buffer (20 mM HEPES, 150 mM NaCl,0.02% NaN₃, pH 7.4) and once with Reaction Buffer (20 mM HEPES, 150 mMNaCl, 5 mM MnCl₂, 5 mM MgCl₂, 0.02% NaN₃, pH 7.4) then concentrated to2.0 mL using a Centricon centrifugal filter, 5 KDa MWCO.

The hGH-TTT mutant was combined with UDP-GalNAc (1.38 micromoles, 0.90mg) and GalNAc-T2 (0.12 mL, 120 mU). The reaction was incubated at 32°C. with gentle shaking for 19 hours. The reaction was analyzed byMALDI-MS and partial addition of GalNAc to the hGH-TTT mutant wasobserved (approximately 40%). CMP-SA-PEG-30K (16 mg, 0.533 micromoles)and ST6GalNAcl (0.375 mL, 375 mU) were added to the reaction mixture tobring the total volume to 2.85 mL. The reaction was incubated at 32° C.with gentle shaking for 22 h. The reaction was monitored by SDS PAGE at0 h and 22 h. The extent of reaction was determined by SDS-PAGE gel. Theproduct, hGH-(TTT)-GalNAc-SA-PEG-30 KDa, was purified using SP Sepharoseand analyzed by SDS-PAGE. Very low yield of the desiredhGH-(TTT)-GalNAc-SA-PEG-30 KDa was observed.

13.3 Preparation of hGH-(PTQGAMP)-GalNAc-SA-PEG-30 KDa

The PTQGAMP mutant was readily glycosylated with UDP-GalNAc and GalNAcT2, then GlycoPEGylated using CMP-SA-PEG-30 KDa and ST6GalNAcl on 10 mgscale to yield 1.45 mg of purified hGH-(PTQGAMP)-GalNAc-SA-PEG-30 KDa.Peptide mapping experiments (trypsin digest) located the GalNAc on thetrypsin T12 peptide (L129-K141) containing the PTQGAMP mutation.

The hGH-PTQGAMP-mutant (4.55 mL, 10.0 mg, 0.45 micromoles) was bufferexchanged twice with 15 mL of Washing Buffer (20 mM HEPES, 150 mM NaCl,0.02% NaN₃, pH 7.4) and once with Reaction Buffer (20 mM HEPES, 150 mMNaCl, 5 mM MnCl₂, 5 mM MgCl₂, 0.02% NaN₃, pH 7.4) then concentrated to 3mL using a Centricon centrifugal filter, 5 KDa MWCO.

The hGH-PTQGAMP mutant was combined with UDP-GalNAc (2.26 micromoles,1.47 mg) and GalNAc-T2 (0.1 mL, 100 mU). The reaction was incubated at32° C. with gentle shaking for 22 hours. The reaction was analyzed byMALDI-MS and complete addition of GalNAc to the hGH-PTQGAMP mutant wasobserved. CMP-SA-PEG-30K (27 mg, 0.9 micromoles) and ST6GalNAcl (0.350mL, 350 mU) were added to the reaction mixture to bring the total volumeto 3.4 mL. The reaction was incubated at 32° C. with gentle shaking for24 h. The reaction was monitored by SDS PAGE at 0 hours and 16.5 hours.The extent of reaction was determined by SDS-PAGE gel. The product,hGH-(PTQGAMP)-GalNAc-SA-PEG-30 KDa, was purified using SP SepharoseandSEC (Superdex 200) chromatography and then formulated. The final productwas analyzed by MALDI, peptide map and SDS-PAGE (silver stain). Proteinwas determined by BCA vs. BSA standard. The overall isolated yield (1.45mg) was 12.5% based on protein.

Example 14

This example sets forth the preparation of a GM-CSF PEG glycoconjugateof the invention.

14.1 Preparation of (PEG(20K)-SA-Gal-GalNAc)₂-GM-CSF andPEG(20k)-SA-Gal-GalNAc-GM-CSF

GM-CSF (1 mg) was dissolved in 25 mM MES buffer (1 mL) (pH 6.0, 0.005%NaN₃), then UDP-GalNAc (1 mg), GalNAc-T₂ (200 μL, 0.38 U/mL, 0.076 U),100 mM MnCl₂ (80 μL) were added. The resulting mixture was incubated atroom temperature for 72 h. MALDI indicated GalNAc2-GM-CSF was formed.

UDP-Gal (6 mg, 9.8 mmol), core-1-Gal-T₁ (0.5 U/mL, 80 μL), CMP-SA-PEG(20 kilodalton) (6 mg, 0.3 μmol), α-(O)-sialyltransferase (1 U/mL, 120μL), 100 mM MnCl₂ (50 μL) were added. The resulting mixture was slowlyrotated at 32° C. for 48 h. The reaction mixture was centrifuged at 2rpm for 5 min. The protein solution was taken. The remain resin wasmixed with 1 mL 25 mM MES buffer (pH 6.0) and vibrated for 30 sec. Thesuspension was concentrated in again; the protein solutions werecombined and concentrated to 200 mcL. HPLC Purification providedglyco-PEG-ylated GM-CSF.

Example 15

An O-linked glycosylation site similar to that of interferon alpha-2 canbe incorporated into any interferon alpha protein at the same relativeposition. This can be performed by aligning the amino acid sequence ofinterest with the IFN-alpha-2b sequence (10-20 amino acids long) andmodifying the amino acid sequence to incorporate the glycosylation site.Mutation with any amino acid, deletion or insertion can be used tocreate the site. Exemplary mutants maintain as high an homology aspossible with the IFN-alpha-2 sequence in this region with an emphasison the T at position 106 (shown below in bold). An example of how thisis performed is shown below.

Alignments of Interferon alpha's in the NCBI Protein Database GI# AA# AASequence Name IFN-a-2β  1 CVIQGVGVTETPLMKEDSIL  20 (SEQ ID NO:X)  124449 98 .................... 117 IFN-alpha 2 (a,b, c) 20178265 99....E...E.....N..... 118 IFN-alpha 14   124453 99 ....E...E.....N.....118 IFN-alpha 10   585316 99 ....E...E.....N..... 118 IFN-alpha 17  124442 99 ....E...E.....N..F.. 118 IFN-alpha 7   124438 99....E...E.....NV.... 118 IFN-alpha 4   417188 99 ..M.E...I.S...Y.....118 IFN-alpha 8 20178289 99 ....E...E.....NV.... 118 IFN-alpha 21  124457 99 .MM.E...ED....NV.... 118 IFN-alpha 5   124463 99..T.E...E.IA..N..... 118 IFN-alpha 16   124460 99 ..M.E.W.GG....N.....118 IFN-alpha 6   124455 99 ..M.EER.G.....NA.... 118 IFN-alpha 1/13

Glycosylation/Glyco-PEG-ylation occurs at T¹⁰⁶ (IFN-alpha-2). Proteinnumbering begins with the first amino acid after removal of the proteinleader sequence of the naturally expressed pre-pro form.

Interferon alpha mutations to introduce O-Linked Glycosylation Sites inIFN-alpha's that lack this site.

GI# AA# AA Sequence Name IFN-a-2β  1 CVIQGVGVTETPLMKEDSIL  20 (SEQ IDNO:X)   124449 98 .................... 117 IFN-alpha 2 (a,b, c) 2017826599 ....E...T.....N..... 118 IFN-alpha 14 (E¹⁰⁷T) 20178265 99....G...T.....N..... 118 IFN-alpha 14 (E¹⁰³G; E¹⁰⁷T)   124453 99....E...T.....N..... 118 IFN-alpha 10 (E¹⁰⁷T) ∩ 124453 99....G...T.....N..... 118 IFN-alpha 10 (E¹⁰³G; E¹⁰⁷T) ∩ 585316 99....E..MT.....N..... 118 IFN-alpha 17 (E¹⁰⁷T)   585316 99....E..VT.....N..... 118 IFN-alpha 17 (ME¹⁰⁷VT)   585316 99....G..MT.....N..... 118 IFN-alpha 17 (E¹⁰³G; E¹⁰⁷T)   124442 99....E...T.....N..F.. 118 IFN-alpha 7 (E¹⁰⁷T)   124442 99....G...T.....N..F.. 118 IFN-alpha 7 (E¹⁰³G; E¹⁰⁷T)   124438 99....E...T.....NV.... 118 IFN-alpha 4 (E¹⁰⁷T)   124438 99....G...T.....NV.... 118 IFN-alpha 4 (E¹⁰³G; E¹⁰⁷T)   417188 99..M.E...T.S...Y..... 118 IFN-alpha 8 (I¹⁰⁷T)   417188 99..M.G...T.S...T..... 118 IFN-alpha 8 (E¹⁰³G; I¹⁰⁷T) 20178289 99....E...T.....NV.... 118 IFN-alpha 21 (E¹⁰⁷T) 20178289 99...G...T.....NV.... 118 IFN-alpha 21 (E¹⁰³G; E¹⁰⁷T)   124457 99.MM.E...TD....NV.... 118 IFN-alpha 5 (E¹⁰⁷T)   124457 99.MM.E...TE....NV.... 118 IFN-alpha 5 (ED¹⁰⁸TE)   124457 99.MM.G...TD....NV.... 118 IFN-alpha 5 (E¹⁰³G; E¹⁰⁷T)   124463 99..T.E...T.IP..N..... 118 IFN-alpha 16 (E¹⁰⁷T; A¹¹⁰P)   124463 99..T.E...T.TP..N..... 118 IFN-alpha 16 (E¹⁰⁷T; IA¹¹⁰TP)   124463 99..T.G...T.TP..N..... 118 IFN-alpha 16 (E¹⁰³G; E¹⁰⁷T; IA¹¹⁰TP)   12446099 ..M.E.W.TG...N..... 118 IFN-alpha 6 (G¹⁰⁷T)   124460 99..M.E.G.TG....N..... 118 IFN-alpha 6 (W¹⁰⁵G; G¹⁰⁷T)   124460 99..M.G.G.TE....N..... 118 IFN-alpha 6 (E103G; W¹⁰⁵G; GG¹⁰⁸TE)   124455 99..M.EER.T.....NA.... 118 IFN-alpha 1/13 (G¹⁰⁷T)   124455 99..M.EEG.T.....NA.... 118 IFN-alpha 1/13 (R¹⁰⁵G; G¹⁰⁷T)   124455 99..M.GVG.T.....NA.... 118 IFN-alpha 1/13 (EER¹⁰⁵GVG; G¹⁰⁷T)The GI numbers in the above table, except the first number 124449, referto those of the unmodified wild-type proteins.

The O-linked glycosylation site can be created in any interferon alphaisoform by placing a T or S at the appropriate amino acid site as shownabove. The substitution is T as shown in the above table. The amino acidsequences between the various interferon alpha forms are similar. Anyamino acid mutation, insertion, deletion can be made in this region aslong as the T or S is at the appropriate position forglycosylation/glyco-PEG-ylation relative to P¹⁰⁹ (IFN-alpha-2) in thealignment sequence shown above.

While this invention has been disclosed with reference to specificembodiments, it is apparent that other embodiments and variations ofthis invention may be devised by others skilled in the art withoutdeparting from the true spirit and scope of the invention.

All patents, patent applications, and other publications cited in thisapplication are incorporated by reference in the entirety.

1. A method of separating a peptide conjugate from a reaction mixture,said method comprising: (a) contacting said reaction mixture with ahydrophobic interaction chromatography medium; and (b) collecting saidpeptide conjugate from said hydrophobic interaction chromatographymedium, thereby separating said peptide conjugate from said reactionmixture.
 2. The method according to claim 1, wherein said peptideconjugate includes a modifying group which is poly(ethylene glycol). 3.The method according to claim 2, wherein said modifying group isattached to said peptide of said peptide conjugate through an intactglycosyl linking group.
 4. A conjugate between a peptide and a modifyinggroup, wherein said conjugate is obtained by a method comprising: (a)contacting a reaction mixture comprising said conjugate with ahydrophobic interaction chromatography medium; and (b) collecting saidconjugate, thereby separating said peptide conjugate from said reactionmixture.
 5. The conjugate according to claim 4, wherein said modifyinggroup is poly(ethylene glycol).
 6. The method according to claim 5,wherein said modifying group is attached to said peptide through anintact glycosyl linking group.